Feasibility, potency, and safety of growing human mesenchymal stem cells in space for clinical application

Peng Huang; Athena L. Russell; Rebecca Lefavor; Nisha C. Durand; Elle James; Larry Harvey; Cuiping Zhang; Stefanie Countryman; Louis Stodieck; Abba C. Zubair

doi:10.1038/s41526-020-0106-z

Feasibility, potency, and safety of growing human mesenchymal stem cells in space for clinical application

Huang, Peng; Russell, Athena L.; Lefavor, Rebecca; Durand, Nisha C.; James, Elle; Harvey, Larry; Zhang, Cuiping; Countryman, Stefanie; Stodieck, Louis; Zubair, Abba C. 2020-06-01 00:00:00 www.nature.com/npjmgrav ARTICLE OPEN Feasibility, potency, and safety of growing human mesenchymal stem cells in space for clinical application 1,2 1,2 1,2 1,2 1,2 3 1,2 Peng Huang , Athena L. Russell , Rebecca Lefavor , Nisha C. Durand , Elle James , Larry Harvey , Cuiping Zhang , 4 4 1,2 Stefanie Countryman , Louis Stodieck and Abba C. Zubair Growing stem cells on Earth is very challenging and limited to a few population doublings. The standard two-dimensional (2D) culture environment is an unnatural condition for cell growth. Therefore, culturing stem cells aboard the International Space Station (ISS) under a microgravity environment may provide a more natural three-dimensional environment for stem cell expansion and organ development. In this study, human-derived mesenchymal stem cells (MSCs) grown in space were evaluated to determine their potential use for future clinical applications on Earth and during long-term spaceﬂight. MSCs were ﬂown in Plate Habitats for transportation to the ISS. The MSCs were imaged every 24–48 h and harvested at 7 and 14 days. Conditioned media samples were frozen at −80 °C and cells were either cryopreserved in 5% dimethyl sulfoxide, RNAprotect, or paraformaldehyde. After return to Earth, MSCs were characterized to establish their identity and cell cycle status. In addition, cell proliferation, differentiation, cytokines, and growth factors’ secretion were assessed. To evaluate the risk of malignant transformation, the space-grown MSCs were subjected to chromosomal, DNA damage, and tumorigenicity assays. We found that microgravity had signiﬁcant impact on the MSC capacity to secrete cytokines and growth factors. They appeared to be more potent in terms of immunosuppressive capacity compared to their identical ground control. Chromosomal, DNA damage, and tumorigenicity assays showed no evidence of malignant transformation. Therefore, it is feasible and potentially safe to grow MSCs aboard the ISS for potential future clinical applications. npj Microgravity (2020) 6:16 ; https://doi.org/10.1038/s41526-020-0106-z INTRODUCTION on Earth. On average astronauts on the International Space Station (ISS) received about 10 times more radiation than people on According to the US Census Bureau’s Projections, by the year 2040, Earth . Therefore, aging and radiation-induced organ damage is a 25% of the US population will be 65 years and older . Therefore, the health hazard, especially in long duration spaceﬂight, and is a incidence of age-related diseases such as dementia, neurodegenera- major concern when humans attempt to colonize other planets. tive diseases, stroke, and cancer will continue to increase. Modern Since organ donors will be very scarce in space, the need for medicine is evolving to address the challenges of this rapidly regenerative therapies in this setting will become paramount. increasing public health burden. Currently, the most effective method Mesenchymal stem cells (MSCs) are multipotent cells known to to treat diseased and/or failed organs is organ replacement through modulate immune cell activation and induce tissue repair and transplantation. However, there are not enough donor organs promote regeneration . MSCs are ﬁbroblast-like adherent cells available to meet the needs of the growing number of patients on that primarily differentiate into osteocytes, chondrocytes, and the waiting list. Expansion of stem cells for regenerative medicine 8–10 adipocytes . They secrete a broad range of cytokines and therapies remains a challenge . With the extraordinary growth of growth factors that promote regeneration of other tissue-resident regenerative medicine, the demand for stem cells is greatly stem cells, such as skeletal, hematopoietic, and neural stem cells. increasing. Stem cells in general are quiescent and maintain tight MSCs are identiﬁed in vitro by their plastic adherence and control of their numbers within tissues. It is generally believed that expression of surface markers, such as STRO-1, CD73, CD90, stem cells constitute <1% of cells in any given organ . CD105, CD271, and CD146, as well as lack of expression of mature When induced to proliferate in culture, stem cells can activate hematopoietic lineage markers such as those expressed by B, T, pathways involved in differentiation or senescence and may lose and myeloid cells. They have been used safely in preclinical and their stemness phenotypes after prolonged ex vivo propagation . clinical studies to modulate inﬂammation and induce tissue repair, Most growth media can barely expand stem cells, and if they 11–14 although the mechanisms are not completely understood .We succeed, it tends to be a slow process. Expansion of the large recently showed that interleukin-6 (IL-6) and vascular endothelial numbers of stem cells needed for clinical applications is growth factor play critical roles in MSC-induced neuro-regenera- challenging, and standard culture medium formulations are tion and anti-inﬂammation . The involvement of IL-6 is perplex- limited in their ability to recapitulate physiological growth ing since IL-6 has been well characterized as a pro-inﬂammatory conditions . Since the force of gravity is pervasive and affects cytokine. virtually all aspects of human physiology, we assessed the MSCs are known to respond to applied- or cell-generated feasibility, potency, and safety of expanding stem cells under mechanical forces . MSC’s role in mechano-transduction mechan- true microgravity conditions for human application. isms associated with bone repair and regeneration has been In microgravity, time can appear to pass faster and as a result extensively studied on Earth and to a lesser extent in real and humans in space age slower because of the time-dilation effect . 17–20 Also, exposure to cosmic radiation is much higher in space than simulated microgravity . Unfortunately, it has been 1 2 3 Department of Laboratory Medicine and Pathology, Mayo Clinic, Jacksonville, FL, USA. Center for Regenerative Medicine, Mayo Clinic, Jacksonville, FL, USA. Center for Applied Space Technologies, Merritt Island, FL, USA. BioServe Space Technologies, University of Colorado Boulder, Boulder, CO, USA. email: Zubair.Abba@Mayo.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; P. Huang et al. challenging to compare the data generated from these experi- could be attributed to the freezing technique, which did not ments because of variation in MSC culture methods, the extent of involve the use of controlled rate freezing. To assess sterility, gram population doubling or passaging, use of different growth factors, stain and cultures were performed and found to be negative for tissue source, and donor-to-donor variation. Also, many studies both sMSCs and gMSCs. Following the immunophenotypic criteria utilized MSC cell lines, while others used primary cultures. As such, set forth by the International Society for Cellular Therapy , both the data generated using each of these highly variable sMSCs and gMSCs were stained and analyzed for expression of approaches may not be comparable. CD73, CD90, and CD105, and lack of CD34, CD11b, CD19, HLA-DR, There are conﬂicting reports regarding MSC proliferation and CD45 expression by ﬂow cytometry. Our analysis showed that 21–23 characteristics under simulated microgravity .Thiscan be both sMSCs and gMSCs had comparable expression of triple- attributed to varying experimental approaches, which include positive markers (CD73+, CD90+, CD105+) and absence of different types of microgravity simulators, the use of micro-carriers, mature hematopoietic lineage markers (Fig. 2 and Table 1). levels of oxygenation, and culture media. There are many types of Fluorescence-activated cell sorting sequential gating strategy has microgravity simulators; the most common is a rotating wall vessel. been provided as Supplementary Fig. 1. The rotating wall vessel may not provide true microgravity To assess MSC proliferation potential after space travel, sMSCs conditions, rather it works by altering the direction of gravity with and gMSCs were further cultured to quantify their relative growth respect to the sample over time, or generating rotational centrifugal 18,19 rates. We determined that both sMSCs and gMSCs have forces counteracting the gravitational force . Therefore, space- comparable proliferation rates (Fig. 3). ﬂight experiments like our own are greatly needed to further understand the impacts of real microgravity on MSCs. Despite the limited value of the data generated from simulated Spaceﬂight environment induces changes to cell cycle checkpoint microgravity experiments mainly using animal MSCs, it can be gene expression inferred from these studies that microgravity inhibits the growth Cells preserved in RNAprotect were subjected to RNA isolation and of MSCs by arresting the cells in the G0/G1 phase of the cell cycle real-time PCR for cell cycle checkpoint gene expression analysis (Fig. and diminishes the cellular response to growth factor stimulation. 4). Compared to gMSCs, the expression of CDKN2A,aG1/S Also, microgravity limits MSC’s capacity to undergo osteogenic checkpoint inhibitor, was not signiﬁcantly different in sMSCs after differentiation through regulation of RUNX2 activity and enhances 1 or 2 weeks in culture. This suggests that within 7–14 days culture, adipogenic differentiation by upregulating PPARγ2 activity . microgravity had minimal impact on MSC capacity to enter into the The immediate need for stem cells for regenerative therapies is S phase of cell cycle by altering the expression of CDKN2A.E2F1 on Earth. The Food and Drug Administration released stringent protein is another cell cycle checkpoint inhibitor that preferentially guidelines regarding the use of cellular products for therapeutic purposes outlined under Code of Federal Regulations 1271. These binds to retinoblastoma protein pRB in a cell cycle-dependent standards are universal regardless of the source and method used manner, thereby promoting entry into S phase. Our analysis showed to expand the cells. All cell-based products intended for human that E2F1 expression by sMSCs may be decreased in microgravity use must have established critical quality attributes to ensure environment, although this decrease was not signiﬁcant. Polo-like identity, purity, sterility, and potency of the product. Tumorigenic kinase 1 (PLK1) is a serine/threonine-protein kinase that facilitates potential of the culture-expanded cells must also be evaluated. the transition from G2 to M phase of the cell cycle. PLK1 promotes This is paramount considering the radiation exposure risk in space maturation of the centrosome and establishment of the bipolar is several times higher than the risk on Earth. spindle. Our analysis showed that PLK1 expression by MSCs in In this study, we evaluated the feasibility and safety of growing culture was decreased in microgravity environment, but this MSCs forhuman application atthe ISS. We have established the decrease was only signiﬁcant after 2 weeks in culture on the ISS. identity, purity, viability, and sterility of the space-grown cells In summary, it appears in a 7-day culture, microgravity does not compared to ground controls. We have further assessed the signiﬁcantly alter the expression of CDKN2A or E2F1 or PLK1. functional characteristics of the space-grown cells and assessed their However, with a 14-day culture, microgravity appeared to down- tumorigenic potential. Overall, we have established the feasibility and regulate the expression of PLK1. Therefore, microgravity conditions safety of MSCs grown on the ISS for human application. may slow the progression of MSCs at later stages of the cell cycle during longer-term culture, but this does not appear to reduce their overall growth rate relative to ground controls. Further studies will RESULTS be needed to fully validate this ﬁnding. MSCs maintain their phenotype and proliferative characteristics after expansion on ISS MSCs were seeded in BioCell cassettes (1500 cells/cm ) 3 days Prior microgravity exposure does not affect MSC differentiation before launch and kept in Plate Habitats (PHABs) at 37 °C. Cell capacity imaging was initiated at ISS 2 days after launch. Cells were imaged Osteogenic and adipogenic differentiation assays performed in in real time at multiple time points for 2 weeks and images were our lab after the space-grown cells were returned to Earth beamed down to Earth (Fig. 1). The ground control cells were demonstrated that sMSCs and gMSCs retained similar capacities to imaged simultaneously in our lab. Real-time images of MSCs at ISS differentiate into bone and adipose tissues, as shown with Alizarin showed no obvious differences in morphology and proliferation Red S and Oil Red O stains, respectively (Fig. 5a). To further rate when compared with ground controls. MSCs were cryopre- investigate the effect of microgravity exposure on MSC differ- served in 5% dimethyl sulfoxide and kept frozen at <−95 °C on entiation, we analyzed the expression of genes associated with the ISS until they were returned to Earth using a SpaceX return osteogenic (COL1A1 and RUNX2) and adipogenic (CEBPB) differ- capsule that landed in the Paciﬁc Ocean. Cells were shipped to our entiation by quantitative reverse transcription PCR. Our analysis laboratory at Mayo Clinic Florida within 24 h by FedEx. showed that the expression of the selected genes in space-grown In our lab the space-expanded MSC (sMSCs) were carefully MSCs was statistically similar to ground controls (Fig. 5b). This thawed and we reafﬁrmed their MSC identity, purity, sterility and suggests that MSC expansion for up to 2 weeks in microgravity post-ﬂight proliferation rate compared with the ground control MSCs (gMSCs). Upon thawing, the mean viability of sMSC was environment does not affect their capacity to differentiate into comparable to that of gMSCs (Table 1). This low viability of cells osteoblasts and adipocytes. npj Microgravity (2020) 16 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; P. Huang et al. Fig. 1 Cell culture and real-time imaging. MSCs were seeded and cultured in BioCells cassettes (a) and transported in Plate Habitats (PHABS) (b). Real-time images were taken from ground control and ISS microgravity groups. Imaging started 3 days before launching (L − 3) until 13 days after launching (L + 13) (c). Scale bar: 400 µm. Spaceﬂight environment altered MSC capacity to secrete altered by microgravity environment (Fig. 6). Platelet-derived cytokines and growth factors growth factor-AA subunit (PDGF-AA), a potent growth factor known to induce regeneration and tissue repair, was signiﬁcantly Conditioned media from sMSC and gMSC cultures were evaluated increased and sCD40L, a powerful immunosuppressant, was for cytokine and growth factor secretion proﬁles. Sixty-ﬁve decreased signiﬁcantly after 1 week in microgravity. IL-10, a cytokines and growth factors were evaluated. Secretion of six potent T-helper type 2 (Th2) cytokine with tolerance-inducing cytokines and growth factors were identiﬁed to be signiﬁcantly properties, macrophage chemotactic protein-3 (MCP-3), and Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 16 P. Huang et al. Table 1. Summary of MSC growth characteristics. Pre-ﬂight Post ﬂight Post ﬂight Post ﬂight Post ﬂight Duration in culture (week) 1 1 1 2 2 Gravity Ground Ground ISS Ground ISS Identity % (CD73+, CD90+, CD105+) 89.19 80.2 80.9 88.9 78.6 Viability %, mean ± SD 96 ± 1 60.5 ± 14.9 53.1 ± 12.7 67.4 ± 5.1 54.3 ± 20.2 Mean cell count × 10 per BioCell 0.2–0.5 4.25 ± 2.01 5.12 ± 1.21 5.73 ± 1.80 5.11 ± 2.24 Sterility (Gram stain and culture) Negative Negative Negative Negative Negative Fig. 2 Establishing MSC identity. MSCs thawed from frozen BioCells after 1 week (a–d) and 2 weeks in culture at ISS were tripled stained with CD73, CD90, and CD105 antibodies and then analyzed using ﬂow cytometry. a, c, e, g were from ground control samples. b, d, f, h were from ISS microgravity samples. R7 gate was used to select double positive for CD90 and CD73. R2 gate was applied out of R7 gate to demonstrate triple-positive cells for CD73, CD90, and CD105 (n = 3). stromal cell-derived factor-1α/β (SDF-1α/β), a powerful chemokine evident by less luminescence signal and therefore less amount known to recruit stem cells and immune cells to sites of injury, of ATP in the co-cultured wells. This observation is not due to were signiﬁcantly decreased, while IFN-γ-inducible protein (IP-10) differential proliferation rates of MSCs as two immunomodulation was signiﬁcantly increased after 2 weeks in microgravity environ- potency assays using MSCs from the same donor, with one assay ment relative to ground controls. These observations suggest that involving irradiated non-proliferating MSCs and the other not the effect of microgravity on the secretion of certain cytokines and irradiated (proliferating), yield the same result (see Supplementary growth factors by MSCs is dependent on the duration of exposure Fig. 2). to microgravity conditions. Growing MSCs at the ISS for up to 2 weeks does not compromise Microgravity environment enhanced the immunosuppressive genomic integrity capacity of MSCs To further assess the safety of growing MSCs in space under To evaluate the immunosuppressive capacity of sMSCs compared microgravity conditions, we determined the presence and extent to gMSCs towards peripheral blood mononuclear cells (PBMCs) of DNA damage. This determination was made by visualization in vitro, MSCs were incubated with phytohemagglutinin (PHA)- and subsequent quantiﬁcation of the phosphorylation of histone stimulated PBMCs (Fig. 7). The relative luminescence units (RLUs) variant γH2A.X at serine 139, in the nuclear compartment of cells. from MSCs and PHA-P control wells were subtracted from the RLU Phosphorylation of H2A.X at S139 is one of the initial signaling measurements from experimental wells to correct for ATP events occurring in cells in response to DNA double-strand breaks produced by MSC metabolism. RLU measurements from the wells (DSBs) , and is therefore a well-established marker for DNA containing PBMCs and PHA-P served as a positive control, damage . representative of the maximum PBMC proliferation. This T cell Using immunoﬂuorescence, we could not detect pS.139-γH2A.X proliferation assay was assessed by the amount of ATP content, in the nuclei of cells from any of the experimental conditions which was proportional to the number of metabolically active evaluated; “Ground-1 week” (Fig. 8a1), “Space-1 week” (Fig. 8a7), cells. Rapamycin served as a positive control and could be “Ground-2 weeks” (Fig. 8a13), and “Space-2 weeks” (Fig. 8a19). As observed to suppress the PHA-stimulated T cell proliferation. a positive control, a group of cells from each experimental Compared to gMSCs, sMSCs cultured for 1 or 2 weeks in condition was stimulated with 100 μM etoposide (an inducer of microgravity were signiﬁcantly more immunosuppressive as DNA DSB) for 2 h, and the presence of pS.139-γH2A.X in the npj Microgravity (2020) 16 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA P. Huang et al. Fig. 3 Post-ﬂight MSC proliferation analyses. a MSCs were cultured in triplicates after 1- or 2-week expansion in the ISS environment and on Earth in our laboratory. Cells were seeded into a 96-well plate and stained with IncuCyte Nuclight red to track cell proliferation by nucleus count. Real-time images were acquired by IncuCyte S3 at 2-h interval up to 48 h. Post ﬂight, the ISS-expanded cells proliferated at comparable rate with ground control cells. Statistics determined by one-way ANOVA (n = 3). Error bars represent standard deviation (SD) (n = 3, P > 0.05). b Similarly, assessment of population doubling in longer-term cultures of 1-week ISS-expanded MSCs and the corresponding ground control showed no statistically signiﬁcant difference in growth rate (two-sample Wilcoxon’s rank-sum (Mann–Whitney) test, P = 0.68). Error bars represent standard deviation (SD). nucleus of these cells was readily detectable (Fig. 8a4, 10, 16, 22). stem cells under microgravity conditions for human regenerative For each experimental condition, there was a signiﬁcant difference medicine applications. We chose MSCs because they are the most in the nuclear content of pS.139-γH2A.X between cells treated common type of stem/stromal cell used in clinical trials involving with etoposide and untreated cells (compare Fig. 8a1 with a4, a7 cell therapy. A quick query on clinical trials.gov revealed over 10,000 registered clinical trials involving MSC therapy. Our study is with a10, a13 with a16, and a19 with a22). Similarly as shown in Fig. 8a, quantiﬁcation of pS.139-γH2A.X unique because it involves the use of primary cultures of human MSCs rather than cell lines. Most of the previous reports using ﬂuorescence intensity (Fig. 8b) present in the nuclei of cells MSCs in microgravity conditions were based on human MSC cell showed a signiﬁcant difference between the etoposide-treated lines or cultured animal-derived MSCs. We therefore believe our and -untreated cells in each experimental condition. For all study ﬁndings reﬂect as close to reality in space as is practically conditions assayed, the percentage of pS.139-γH2A.X in the feasible to studying humans under microgravity. untreated cells was <4% when compared to their etoposide- The ﬁrst step towards establishing the clinical utility and safety treated counterparts normalized to 100%. Most importantly, of space-grown cells is ensuring the phenotypic identity of the quantiﬁcation analysis showed that there were no signiﬁcant cells was not changed during growth at ISS under microgravity differences among the four experimental conditions, with very conditions. Our evaluation showed that MSCs maintained their similar percentages of pS.139-γH2A.X reported for each one: physical identity and proliferative characteristics aboard ISS. In “Ground-1 week”—3.7%, “Space-1 week”—3.4%, “Ground- addition, these physical attributes were maintained after they 2 weeks”—3.3%, and “Space-2 weeks”—3.1% (Fig. 8b). were returned to Earth and further expanded under standard These data demonstrate that the MSC nuclear content of gravity conditions in our lab. We could not ﬁnd previous studies pS.139-γH2A.X, and therefore associated DNA DSBs, is not affected that evaluated MSC surface marker identity in real microgravity. by growth in microgravity conditions for up to 2 weeks. However, there were many studies that assessed MSC morpho- In addition, chromosomal studies were performed using logical characteristics under simulated microgravity. Luna et al. conﬂuent cultures of sMSCs and gMSCs. Karyotyping using reported that MSCs responded to clinorotation by adopting a standard G banding method showed no chromosomal abnorm- more rounded, less-spread morphology. alities (Fig. 9). It was challenging to accurately evaluate MSC proliferation rate in real time while in culture at ISS. Resources at ISS and crew time MSCs showed no evidence of tumorigenic transformation after a were the main limiting factors. We resorted to a series of simple 14-day culture aboard ISS microscopic images. These images were sent to us in near real Although we did not detect any evidence of compromised time. In addition, based on a prior experience with ﬁbroblast cell genomic integrity, there is still concern of tumorigenic transfor- lines, our MSCs had to be seeded on Earth to enable the cells to be mation due to the presence of cosmic radiation that may result in adherent, as gravity is apparently needed for this process. Seeding oncogenic mutation. We therefore performed a tumorigenicity the MSCs at ISS under microgravity may have led to non- assay to evaluate both sMSC and gMSC tumorigenic potential at 1- adherence and subsequent cell death. We opted to pre-seed the and 2-week cultures. HT-1080 was used as a positive control cell MSCs on Earth. Post-ﬂight MSC count and viability were impacted line (Fig. 10a) and W138 as negative control cell line (Fig. 10b). by cryopreservation method. Overall, our assessment was that the Neither sMSCs nor gMSCs showed evidence of tumor formation cell counts between ISS-expanded and ground control cells were after a 1- or 2-week culture. comparable. Therefore, it is unclear if there is any difference in proliferation rate between the ISS and ground control cells. However, analysis of cell cycle checkpoint gene expression DISCUSSION showed that the spaceﬂight environment after a 14-day culture The focus of this study was to evaluate the feasibility and safety of caused downregulation of PLK1, a late G1/S and G2/M checkpoint using space-grown MSCs for potential future clinical application promoter. This suggests that microgravity could slow MSC on Earth and during long-term spaceﬂight. Many studies have proliferation in longer-term cultures. Further studies will be shown detrimental effects of the absence of gravity on bone needed to fully elucidate this observation. homeostasis and the musculoskeletal system. In this study, we Our studies showed that prior microgravity exposure does not aimed to identify the beneﬁcial effects of microgravity on stem affect subsequent MSC differentiation on Earth. However, previous cell proliferative capacity with the long-term objective of growing studies of MSC grown in microgravity demonstrated a skewing of Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 16 P. Huang et al. Fig. 4 Impact of microgravity on expression of cell cycle checkpoints associated genes. MSCs were culture for 1 and 2 weeks. Cells were harvested and saved in RNAprotect. RNA was later isolated and quantitative reverse transcription PCR (RT-qPCR) analysis was performed as described in the “Methods” section. Expression of cell cycle checkpoint-associated genes: a cyclin-dependent kinase inhibitor 2A (CDKN2A), b transcription factor (E2F1), and c serine/threonine-protein kinase, known as polo-like kinase 1 (PLK1) were evaluated and compared to ground control gMSCs. Gene expression was normalized to GAPDH. Statistics determined by one-way ANOVA (n = 3, *P < 0.05), error bars represent standard deviation (SD). 24,29 their differentiation capacity towards adipogenic lineage . Our that CXCR4, a receptor for SDF-1, is overexpressed in simulated results do not support these reported studies. Possible explana- microgravity. Similarly, Dr. Kearns-Jonker’s group reported that tion as to why our study differs from these reports could be that simulated microgravity induces cardiac progenitor cells (CPCs) to the effects of microgravity on MSC differentiation to adipogenic increase SDF-1a messenger RNA (mRNA) expression and this effect lineage require longer microgravity exposure beyond 2 weeks was more pronounced in adult CPCs . They also used BioCell culture. Further studies will be needed to determine if prolonged Culture Cassette to grow CPCs at ISS and observed similar SDF-1a exposure to microgravity beyond 2 weeks in culture has lasting mRNA overexpression . Although these ﬁndings were based on effects on the MSC differentiation capacity. mRNA transcripts in CPC, while ours measured protein secretion Our studies showed that the spaceﬂight environment altered by bone marrow-derived MSCs, our study appears to contradict MSC capacity to secrete certain cytokines and growth factors and the observation by Dr. Kearns-Jonker’s group. This differential this effect was more enhanced with time. MSC capacity to secrete ﬁnding highlights the fact that the effect of microgravity varies PDGF-AA and IP-10 was signiﬁcantly increased, while MCP-3 signiﬁcantly among different cell types as well as cells at different (CCL7), IL-10, sCD40L, and SDF-1 (CCL12) was signiﬁcantly stages of their life cycles. Also, mRNA expression may not decreased in microgravity environment relative to ground necessarily correlate with protein expression and secretion. More controls. Contrary to our ﬁndings, it was previously reported that studies will be needed to further investigate these observations. microgravity reduces the expression of PDGF-β receptor in rat Our studies showed that microgravity environment enhanced osteoblasts . Another study reported that human endothelial the immunosuppressive capacity of MSCs. It is established that cells in response to lipopolysaccharide stimulation under micro- microgravity environment and space radiation signiﬁcantly affect 31 15 gravity increased the expression of PDGF . MCP-3 and SDF-1 are the immune system . Immune dysregulation in microgravity both potent chemotactic factors known to play signiﬁcant roles in conditions is not yet fully understood. It is known that cytotoxic T immune regulation, bone formation, and recruitment of cell function is diminished and this is correlated with reactivation 42 42 T lymphocytes and monocytes to local inﬂammatory of latent herpesviruses in some crewmembers . Mehta et al. 32–35 responses . Both immunity and bone formation are known showed a direct correlation of plasma cytokine alterations with 36–38 to be altered in microgravity . It has been suggested that MCP viral shedding in speciﬁc crewmembers, and hypothesized that expression is affected by simulated microgravity. Our study spaceﬂight is associated with Th2-type tolerogenic immune addresses the impact of true microgravity on secretion of MCP- responses . On the contrary, both sCD40L, a powerful immuno- 3. Using rat bone marrow-derived MSC, Mitsuhara et al. reported suppressant, and IL-10, a Th2-type cytokine, secretions by sMSCs npj Microgravity (2020) 16 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA P. Huang et al. Fig. 5 Inﬂuence of microgravity on osteogenic and adipogenic differentiation. sMSC and gMSC cultures were stained with Alizarin Red and Oil Red O (a). Each condition was seeded and measure in triplicates. Representative pictures (×100) are shown from post-ﬂight MSC culture in a growth medium or differentiation medium after 2-week culture expansion at ISS or ground (Earth). Quantitative real-time PCR analysis of osteogenic (COL1A1, RunX2) and adipogenic (CEBPB) genes was done after 21 days in differentiation medium (b). RNA from three 1-week and 2-week MSC cultures were used for quantitative PCR analysis. Gene expression was normalized to GAPDH. Statistics determined by one-way ANOVA (n= 3). Error bars represent standard deviation (SD). Scale bar: 100 µm. were signiﬁcantly decreased in our study. In agreement with these and many limitations. Access to ISS is very limited. During the reports, our T cell proliferation assay showed sMSCs to have planning and execution of our study, only SpaceX in the United enhanced immunosuppressive properties via a mechanism that is States could provide the required up mass to ISS as well as the unlikely to be dependent on IL-10 and sCD40L. Our study desired down mass following the conduct of our research. Other opportunities to access ISS and return payloads to Earth are highlights a potential role of MSCs in the mechanism of spaceﬂight-induced immune dysregulation and tolerance. How- currently in development and might soon become available. In ever, further studies will be needed in order to fully understand addition, opportunities for suborbital studies have since become the mechanism of microgravity-induced immunosuppression. available, such as Blue Origin and the Northrup Grumman Cygnus On the ISS, astronauts receive more than ten times the amount capsule now regularly launches to the ISS. We were very limited by of space and cosmic radiation compared to what humans receive the weight and volume of our payload. We had to modify our plan on Earth. This is despite the fact that the ISS orbit does not travel to eliminate media exchanges and minimize the amounts of through the Van Allen radiation belts. In contrast, long duration reagents and supplies to be used due to reduced crew time and spaceﬂight outside the Van Allen radiation belts could be very cold/warm stowage space on ascent to and descent from the ISS. hazardous to humans. This could result in signiﬁcant DNA damage Use of only one donor in our studies is not ideal as we have and genomic instability that may lead to cancer. This is particularly previously reported that MSC characteristics vary signiﬁcantly concerning if stem cells are affected. Our studies showed growing from donor to donor . Crew time is signiﬁcantly limited as they MSCs at ISS for 2 weeks does not compromise genomic integrity are tasked with multiple projects. Automated bioreactors that are or evidence of tumorigenic transformation. now offered by multiple implementation partners are very useful Even though we have successfully demonstrated that MSCs can and can mitigate some of the challenges highlighted, such as crew be safely expanded at ISS, our study faced signiﬁcant challenges time expenditure and complexity of operating equipment at ISS. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 16 P. Huang et al. Fig. 6 Conditioned media from sMSC and gMSC cultures were evaluated for cytokine and growth factor secretion proﬁle. PDGF-AA (a) was increased and sCD40L (i) was decreased signiﬁcantly after 1 week in microgravity. IL-10 (d), MCP-3 (f), and SDF-1α/β (h) were decreased and IP-10 (l) was increased signiﬁcantly in microgravity environment after 2 weeks in microgravity environment relative to ground controls. Statistics determined by Student’s t test (n = 3, *P < 0.05 **P < 0.01). Error bars represent standard deviation (SD). In summary, it is feasible and safe to grow MSCs aboard the ISS. Space-grown MSCs from our study maintained their morphologi- cal and phenotypic characteristics, proliferative capabilities, differentiation potential, and were even more potent in their immunosuppressive abilities compared to ground control MSCs. These ﬁndings are positive indications that MSCs grown in microgravity could be used for future clinical applications. Our studies demonstrate the feasibility and safety of expanding MSCs at ISS for possible clinical application, but more studies are needed to fully validate our ﬁndings. METHODS Experimental design The experiments utilized BioServe’s single-well BioCell, a cell culture spaceﬂight certiﬁed piece of hardware that can effectively support cell culture. The experiment utilized 17 BioCells containing MSCs were ﬂown in PHABs for transportation to ISS. Once on board the ISS, the PHABs were placed inside of BioServe’s SABL unit, which provides 37 °C temperature Fig. 7 Immunomodulation potency assay comparing the immu- control and 5% CO . The experiment also utilized BioServe’s standard nosuppressant capacity of gMSC and sMSC towards peripheral inverted phase contrast microscope for imaging of the cells once on board blood mononuclear cell (PBMC). MSCs were incubated for 72 h with ISS. It also required samples be frozen at <−95 °C once processed and phytohemagglutinin (PHA) stimulated PBMCs at 1:3 ratio of MSCs to remain frozen until return to Earth. MSC cultures were processed at two PBMCs. Luminescence is proportional to the amount of ATP present, time points: at 7 and 14 days. One BioCell was ﬂown speciﬁcally for and the amount of ATP is proportional to the number of imaging, it was imaged every 48 h for 14 days. MSCs were imaged at ×40 metabolically active cells present. Luminescence fold change is and ×100 magniﬁcations using the VUE camera system from within the relative to the positive control (PBMCs with PHA). Each condition microgravity science glovebox. The microscope utilized at the ISS was a was seeded and measured in quintuplicate. Statistics determined by standard phase contrast inverted microscope (Nikon TS100). A high- one-way ANOVA (n = 5, *P < 0.05, **P < 0.005). Error bars represent resolution camera attached to the microscope was able to capture both standard deviation (SD). still and video images once the cultures were removed from the on-orbit incubator and placed on the microscope stage. Live pictures were taken However, they are very expensive and may consume a signiﬁcant for each imaging BioCell periodically and sent to Earth in real time. At the part of the research budget. We believe the cost will come down end of the experiment, 5 ml 20% paraformaldehyde (PFA; Electron once the commercial crew vehicles come online. This will Microscopy Sciences) (ﬁnal concentration is 4%) was added to the imaging signiﬁcantly increase available crew time on board the ISS to BioCells directly to preserve the cells at <−95 °C until return to Earth. complete scientiﬁc studies such as ours. Our spaceﬂight studies Similarly, gMSCs were treated exactly the same as those sMSCs in answered many of our original questions; however, we are now microgravity environment. Even the timing of cell evaluation and cell left with more questions than answers and the desire to return to preservation was approximately maintained. Results of experiments shown ISS to perform more experiments. in Figs. 3 and 5 involved post-ﬂight Earth re-expanded cells. npj Microgravity (2020) 16 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA P. Huang et al. Fig. 8 Genomic integrity analysis of the safety of growing MSC in space. a Bone marrow-derived mesenchymal stem cells (5000 cells/ well) previously grown for 1 week or 2 weeks in space, along with ground control cells were seeded to ibidi µ-Slide 8 Well. Cells were stimulated with 100 μM etoposide as indicated, ﬁxed, and subjected to immunoﬂuorescence analysis to visualize pS139-H2A.X. Scale bars indicate 10 μm. b shows quantitation of the ﬂuorescence intensity of pS139-H2A.X (n = 40 cells). Analysis was conducted using the Image J software. Fluorescence intensities were normalized to the etoposide-treated group for each experimental condition. **** indicates statistical signiﬁcance (<0.0001) determined by one-way ANOVA as compared to cells treated with 100 μM etoposide. Error bars represent standard deviation (SD). fetal bovine serum (FBS; R&D Systems) (designated as Complete Culture 20,21 Medium, CCM) according to a previously reported method . MSCs were expanded to passage 2 and MSC cultures were over 89% pure based on the CD73, CD90, and CD105 co-expression and absence of hematopoietic lineage-speciﬁc markers. Expanded MSCs in 25 ml of CCM were seeded into BioCells at passage 4. BioCells were divided into 1- or 2-week groups. The 1-week group was seeded with 5 × 10 MSCs, while the 2-week group was seeded with 2 × 10 MSCs. Identical BioCells were prepared for ground controls. After launch, no media exchange was performed. At the speciﬁed time points, MSCs were treated and harvested according to the following procedures. A 4.5 ml media sample was pulled into a monovette through a BioCell sample port and frozen at −95 °C directly for cytokine and growth factor analysis. Cells in the BioCell were trypsinized and 1.5 ml of cell sample was mixed with 5 ml of RNAprotect (Qiagen), and then moved to −95 °C for RNA expression analysis. The remaining cells were frozen in cell preserving medium containing 5% dimethyl sulfoxide (Sigma-Aldrich) directly inside the BioCells and stored at −95 °C. Viability post thawing: Immediately after the BioCells were thawed, aliquots were made for further experiments. Cell number and viability per BioCell were evaluated via Trypan blue (0.4%; VWR) and documented. Post-ﬂight real-time proliferation analysis: Six sMSC and gMSC aliquots from 12 BioCells were selected for real-time cell proliferation analysis post ﬂight. Each aliquot was expanded and 2 × 10 cells/well seeded into a 96- well plate. IncuCyte NucLight Red ﬂorescence dye (Essen BioScience) was then added to each well at 1:500 dilution. The 96-well plate-containing cells was loaded into the IncuCyte (Essen Bioscience) with scheduled scanning at 2-h intervals up to 48 h. Automated cells counts from each well were further analyzed using the Prism 8 software (GraphPad). MSC phenotyping by ﬂow cytometry Expanded MSCs were harvested with 0.05% Trypsin (Fisher Scientiﬁc). Cells were pelleted at 300 × g for 5 min, re-suspended in 2 ml of phosphate- buffered saline (PBS) and ﬁltered through a 35-µm nylon-mesh ﬁlter (Fisher Scientiﬁc), pelleted again at 300 × g for 5 min, and re-suspended to a ﬁnal volume of 1 ml of PBS. Filtered cells were dispensed to 100 μl aliquots, incubated with MSC marker antibodies against CD73, CD90, and CD105 (BD Biosciences, Human MSC Analysis Kit, Catalog number 562245, Lot number 5313719, 1:100 diluted in BD Pharmingen™ stain buffer), plus antibodies against negative markers CD34, CD11b, CD19, HLA-DR, and CD45 . Cells were incubated with antibodies in the dark for 20 min at room temperature. Stained cells were resuspended in a volume of 600 μl with the addition of 500 μl of PBS prior to analysis by ﬂow cytometry. Cell cycle analysis RNA was extracted using RNeasy Plus Mini Kit (Qiagen) as was previously reported and was prepared for real-time PCR assays. Three biological samples were selected for each group. TaqMan Gene Expression Assays were purchased from Applied Biosystems: GAPDH (Hs02758991_g1), CDKN2A (Hs00923894_m1), E2F1 (Hs00153451_m1), and PLK1 (Hs00153444_m1). TaqMan Fast Advanced Master Mix (Applied Biosystem) Cell culture and proliferation were used according to the manufacturer’s user guide. Each sample was −ΔΔCT Pre-ﬂight mononuclear cells were isolated from a healthy bone marrow tested in triplicate. GAPDH was used as the internal control and the 2 donor using Histopaque-1077 (Sigma-Aldrich) following density gradient method was used to analyze data. GAPDH is a stable housekeeping gene 45–47 protocols and used to culture expand the MSCs. The MSCs were cultured in both on Earth and under microgravity condition . For statistical α-minimum essential medium (Fisher Scientiﬁc) supplemented with 16.5% analysis, unpaired T tests were carried out using GraphPad Prism 8. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 16 P. Huang et al. Fig. 9 Chromosomal studies were performed using conﬂuent cultures of sMSCs and gMSCs. Twenty cells were analyzed from sMSCs (a) and gMSCs (b) and two of these were karyotyped using standard G banding method. No chromosomal abnormalities were detected. Osteogenic and adipogenic differentiation assays in quintuplicate at 6.6 × 10 cells/well and stored in a humidiﬁed 37 °C, 5% CO incubator for 2 h. Following incubation, fresh, primary PBMCs (Astarte Bone differentiation media (BDM) contained CCM plus 1 nM dexametha- 2 Biologics) were re-suspended in a medium (RPMI 1650 medium, 5% FBS, sone (Sigma-Aldrich), 20 mM β-glycerolphosphate (Sigma-Aldrich), and 1× Glutamax, 0.05% penicillin/streptomycin; Fisher Scientiﬁc) and seeded 50 µg/ml L-ascorbic acid 2-phosphate (Sigma-Aldrich) at a ﬁnal concentra- to wells containing MSCs at 2 × 10 cells/well, resulting in a 1:3 ratio of tion. Fat differentiation media (FDM) contained CCM plus 0.5 µM MSCs to PBMCs. PHA-P (Sigma-Aldrich), a mitogen that activates dexamethasone, 0.5 µM isobutylmethylxanthine (Sigma-Aldrich) and proliferation of T cells, was added to all wells containing MSCs at a 50 µM indomethacin (Sigma-Aldrich) at ﬁnal concentration. MSCs were concentration of 10 µg/ml. Additional control conditions were included, seeded at 1 × 10 cells/well in 6-well plates. Cultures were propagated in which contained PBMCs only, PBMCs with PHA-P, and PBMCs with PHA-P CCM until MSC reached 100% conﬂuence. CCM was then replaced with and 10 µg/ml rapamycin (InvivoGen), an IL-2 inhibitor to serve as a BDM and FDM for induction of bone differentiation and fat differentiation. functional control of immunosuppression. The plates were incubated in a Cells were washed with PBS and differentiation media replaced every humidiﬁed 37 °C, 5% CO incubator for 72 h. Following 72 h of incubation, 3 days for 21 days. After 21 days, media were aspirated and cells were the plates were removed and the quantity of ATP in each well was washed with PBS. Two milliliters of 10% formalin (Fisher Scientiﬁc) was measured via luminescence using CellTiter-Glo Luminescent Cell Viability added to each well and cells were incubated for 1 h at room temperature. Kit (Promega), which implements a thermostable luciferase that produces Formalin was aspirated and bone differentiation wells were washed with luminescent signal proportional to the amount of ATP present in the deionized (DI) water, while fat differentiation wells were washed with PBS. sample. The RLUs from MSC and PHA-P control wells were subtracted from One percent Alizarin Red S (Sigma-Aldrich) in DI water was used to stain the RLU measurements from experimental wells to correct for ATP cells for osteogenesis. For adipogenesis staining, ﬁrst 0.5% Oil Red O produced by MSC metabolism. RLU measurements from the wells (Sigma-Aldrich) in isopropyl alcohol was diluted with PBS to make a 0.3% containing PBMCs and PHA-P served as a positive control, representative working solution. Cells were then stained for adipogenesis with 0.3% Oil of the maximum PBMC proliferation. The normalized data was presented Red O. After 20 min at room temperature, the solution was aspirated and as a fold change in comparison to the positive control condition (PBMCs cells were washed with DI water and PBS until wash ﬂuid became clear. with PHA-P). Statistical signiﬁcance was determined by one-way analysis of Each condition was seeded and measure in quintuplicates. Stained cells variance, comparing the means of each condition. were then imaged using Olympus inverted microscope at ×100 magniﬁcation. Chromosomal analysis Chromosomal karyotype was prepared from adherent MSC in a T25 ﬂask Quantitative real-time PCR grown to conﬂuence. At this point, 25 µl of 1 µg/ml colcemid solution RNA from MSCs that has been induced under osteogenic and adipogenic (Sigma-Aldrich) per 2 ml of media was added to the ﬂask and cells were differentiation was extracted using RNeasy Plus Mini Kit as was previously incubated at 37 °C with 5% CO ,5% O , and 90% N for 16–22 h. The 2 2 2 reported and then prepared for real-time PCR assays. Quantitative real- medium was removed from the ﬂask and placed in a 15 ml centrifuge tube time PCR analysis of osteogenic (COL1A1, Hs00164004_m1; RunX2, and TrypLE Express was used to dissociate the cells. Harvested cells were Hs00231692_m1) and adipogenic (CEBPB, Hs00270923_s1) genes was added to the 15 ml centrifuge tube and centrifuged at 300 × g for 8 min. done after MSCs have been exposed in a differentiation medium for The medium was removed and the pellet was re-suspended in 10 ml of 21 days. 50:50 hypotonic solution (potassium chloride 0.075 M:sodium citrate 0.8%) and incubated at RT for 20–30 min. Two milliliters of 3:1 ﬁxative (methanol: Cytokines and growth factor analysis glacial acetic acid) was added and cells were centrifuged. Pelleted cells Cell culture media collected directly from BioCells on the ISS were thawed were re-suspended in 10 ml of 3:1 ﬁxative (methanol:glacial acetic acid). and aliquoted after we received the payload on Earth. Conditioned Centrifugation and addition of ﬁxative was repeated two more times. Cells medium from three BioCells per time point (1 or 2 weeks in culture) were were dropped on pretreated microscope slides at 25 °C and 65% relative aliquoted into 200 µl samples. Secreted cytokines and growth factors were humidity. Twenty cells were analyzed and two of these were karyotyped analyzed using the Human Cytokine/Chemokine 65-Plex Panel (HD65) (Eve using standard G banding method. Technologies). Each sample was analyzed in duplicate, and averaged to represent the cytokine/chemokine value. Student’s t test was used to DNA damage assay compare signiﬁcance between ground and microgravity (ISS) samples. Bone marrow-derived MSCs (5 × 10 cells/well) were seeded to µ-Slide 8 Details cytokines/chemokines list from Eve Technologies has been Well (ibidi) grown to ~70% conﬂuency and stimulated with 100 μM included in Supplementary Fig. 3. etoposide (Abcam) for 2 h as indicated. Following etoposide treatment, cells were washed three times with PBS and ﬁxed with 4% PFA (37 °C, Immunosuppression assay 15 min). After ﬁxation, cells were washed three times with PBS, Cryopreserved MSCs were thawed in a 37 °C water bath and re-suspended permeabilized with 0.1% Triton-X 100 in PBS for 4 min (RT), and blocked in CCM. In white 96-well plates (Greiner, Kremsmünster) MSCs were seeded with 3% bovine serum albumin and 0.05% Tween-20 in PBS (blocking npj Microgravity (2020) 16 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA P. Huang et al. Fig. 10 Tumorigenicity assay. Cells were seeded on a soft agar to evaluate their tumorigenic potential. Five thousand cells/well of positive control cell line HT-1080 (a) and 67,000 cells/well of negative control cell line W138 (b) were used as control to validate the procedure. Twelve cells/well of MSCs were used for this assay. (c) Ground control and (d) microgravity were cells that were culture expanded for 1 week and (e) ground control and (f) microgravity were cells cultured for 2 weeks on Earth and in microgravity, respectively. solution) for 1 h at RT. Samples were incubated with pS.139-H2A.X 4. Russell, A. L., Lefavor, R., Durand, N., Glover, L. & Zubair, A. C. Modiﬁers of antibody (Cell Signaling Technology) in blocking solution at 1:250 for mesenchymal stem cell quantity and quality. Transfusion 58, 1434–1440 (2018). 24 h at 4 °C, washed ﬁve times with PBS and further incubated with goat 5. European Space Agency. Studying How Time is Perceived in Space. https://phys. anti-rabbit immunoglobulin G (H + L) secondary antibody (Fisher org/news/2019-02-space_1.html (2019). Scientiﬁc) at 1:800 and 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) at 6. Forbes, S. How Much Radiation are ISS Astronauts Exposed To? https://www.forbes. 1:5000 in blocking solution for 2 h at RT. Subsequently, cells were washed com/sites/quora/2018/11/13/how-much-radiation-are-iss-astronauts-exposed-to/ ﬁve times with PBS, mounting media (ibidi) was added to the wells, and #2949f6ee18a9 (2018). images were captured using the EVOS FL Cell Imaging System (Fisher 7. Viswanathan, S. et al. Mesenchymal stem versus stromal cells: International Scientiﬁc). Society for Cell & Gene Therapy (ISCT(R)) Mesenchymal Stromal Cell committee position statement on nomenclature. Cytotherapy 21, 1019–1024 (2019). 8. Li, Z., Hu, X. & Zhong, J. F. Mesenchymal stem cells: characteristics, function, Tumorigenicity assay and application. Stem Cells Int. 2019, https://doi.org/10.1155/2019/8106818 Soft agar assay was used to evaluate tumorigenicity of cells grown in (2019). microgravity as well as ground controls. Human ﬁbrosarcoma cell line HT- 9. Karaoz, E., Cetinalp Demircan, P., Erman, G., Gungorurler, E. & Eker Sariboyaci, A. 1080 (ATCC, CCL-121) and ﬁbroblast cell line WI38 (ATCC, CCL-75) were Comparative analyses of immunosuppressive characteristics of bone-marrow, used as positive and negative controls. Cells were suspended in 2 ml of top Wharton’s jelly, and adipose tissue-derived human mesenchymal stem cells. Turk. agar (0.35% agar in α-minimum essential medium containing 20% FBS) J. Haematol. 34, 213–225 (2017). and then plated on 3 ml of bottom agar (0.5% agar in α-minimum essential 10. Pourrajab, F., Forouzannia, S. K. & Tabatabaee, S. A. Molecular characteristics of medium containing 20% FBS) in a 6-well plate. Colonies were counted after bone marrow mesenchymal stem cells, source of regenerative medicine. Int. J. a 14-day incubation. Cardiol. 163, 125–131 (2013). 11. Keller, C. A. et al. Feasibility, safety, and tolerance of mesenchymal stem cell therapy for obstructive chronic lung allograft dysfunction. Stem Cells Transl. Med. Reporting summary 7, 161–167 (2018). Further information on research design is available in the Nature Research 12. Huang, P. et al. Safety and efﬁcacy of intraventricular delivery of bone marrow- Reporting Summary linked to this article. derived mesenchymal stem cells in Hemorrhagic Stroke Model. Sci. Rep. 9, 5674 (2019). 13. Resnick, I. B. et al. Treatment of severe steroid resistant acute GVHD with DATA AVAILABILITY mesenchymal stromal cells (MSC). Am. J. Blood Res. 3, 225–238 (2013). The authors declare that the data that support the ﬁndings of this study are available 14. Ball, L. M. et al. Multiple infusions of mesenchymal stromal cells induce sustained within the paper. remission in children with steroid-refractory, grade III-IV acute graft-versus-host disease. Br. J. Haematol. 163, 501–509 (2013). 15. Huang, P. et al. Mechanism of mesenchymal stem cell-induced neuron recovery Received: 9 December 2019; Accepted: 7 May 2020; and anti-inﬂammation. Cytotherapy. https://doi.org/10.1016/j.jcyt.2014.05.007 (2014). 16. Chen, J. C., Chua, M., Bellon, R. B. & Jacobs, C. R. Epigenetic changes during mechanically induced osteogenic lineage commitment. J. Biomech. Eng. 137, 020902 (2015). REFERENCES 17. Pao, S. I. et al. Effect of microgravity on the mesenchymal stem cell characteristics 1. Koebnick, C. et al. Sociodemographic characteristics of members of a large, of limbal ﬁbroblasts. J. Chin. Med. Assoc. 80, 595–607 (2017). integrated health care system: comparison with US Census Bureau data. Perm J. 18. Blaber, E. A. et al. Mechanical unloading of bone in microgravity reduces 16,37–41 (2012). mesenchymal and hematopoietic stem cell-mediated tissue regeneration. Stem 2. Chen, C., Hu, Z., Liu, S. & Tseng, H. Emerging trends in regenerative medicine: a Cell Res. 13, 181–201 (2014). scientometric analysis in CiteSpace. Expert Opin. Biol. Ther. 12, 593–608 (2012). 19. Mao, X., Chen, Z., Luo, Q., Zhang, B. & Song, G. Simulated microgravity inhibits the 3. Reya, T., Morrison, S. J., Clarke, M. F. & Weissman, I. L. Stem cells, cancer, and migration of mesenchymal stem cells by remodeling actin cytoskeleton and cancer stem cells. Nature 414, 105–111 (2001). increasing cell stiffness. Cytotechnology 68, 2235–2243 (2016). Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 16 P. Huang et al. 20. Yu, B. et al. Simulated microgravity using a rotary cell culture system promotes 44. Tamimi, Y. et al. FGF19 is a target for FOXC1 regulation in ciliary body-derived chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 cells. Hum. Mol. Genet. 15, 3229–3240 (2006). MAPK pathway. Biochem. Biophys. Res. Commun. 414, 412–418 (2011). 45. Cotrupi, S., Ranzani, D. & Maier, J. A. Impact of modeled microgravity on micro- 21. Wang, N. et al. The simulated microgravity enhances multipotential differentia- vascular endothelial cells. Biochim. Biophys. Acta 1746, 163–168 (2005). tion capacity of bone marrow mesenchymal stem cells. Cytotechnology 66, 46. Thiel, C. S. et al. Stability of gene expression in human T cells in different gravity 119–131 (2014). environments is clustered in chromosomal region 11p15.4. npj Microgravity 3,22 22. Zayzafoon, M., Gathings, W. E. & McDonald, J. M. Modeled microgravity inhibits (2017). osteogenic differentiation of human mesenchymal stem cells and increases 47. Parra, M. et al. Microgravity validation of a novel system for RNA isolation and adipogenesis. Endocrinology 145, 2421–2432 (2004). multiplex quantitative real time PCR analysis of gene expression on the Inter- 23. Mayer-Wagner, S. et al. Simulated microgravity affects chondrogenesis and national Space Station. PLoS ONE 12, e0183480 (2017). hypertrophy of human mesenchymal stem cells. Int. Orthop. 38, 2615–2621 (2014). 24. Zhang, C. et al. Space microgravity drives transdifferentiation of human bone ACKNOWLEDGEMENTS marrow-derived mesenchymal stem cells from osteogenesis to adipogenesis. We acknowledge and thank the Center for the Advancement of Science in Space FASEB J. 32, 4444–4458 (2018). (CASIS), now renamed as National Laboratory, Mayo Clinic Center for Regenerative 25. Dominici, M. et al. Minimal criteria for deﬁning multipotent mesenchymal stromal Medicine, BioServe, and the following members of CAST (Center for the Applied cells. The International Society for Cellular Therapy position statement. Cyto- Space Technology) for their support during the early phase of this project: Maria therapy 8, 315–317 (2006). Peterson, Bob Twiggs, and Richard Snyder, Director, University of Florida Center of 26. Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S. & Bonner, W. M. DNA double- Excellence for Regenerative Health Biotechnology. This project was funded by Center stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. for the Advancement of Science in Space (Grant no. GA-2014-127 to A.C.Z.). Chem. 273, 5858–5868 (1998). 27. Yuan, J., Adamski, R. & Chen, J. Focus on histone variant H2AX: to be or not to be. FEBS Lett. 584, 3717–3724 (2010). AUTHOR CONTRIBUTIONS 28. Luna, C., Yew, A. G. & Hsieh, A. H. Effects of angular frequency during clinor- A.C.Z. conceived the idea and designed the study, supervised the work, and wrote otation on mesenchymal stem cell morphology and migration. npj Microgravity 1, the manuscript. P.H. was involved in planning, study design validation, data 15007 (2015). collection, and contributed to writing the manuscript. A.L.R., R.L., N.C.D., E.J., and C.Z. 29. Xue, L., Li, Y. & Chen, J. Duration of simulated microgravity affects the differ- were involved in data collection and contributed to writing the manuscript. P.H. and entiation of mesenchymal stem cells. Mol. Med. Rep. 15, 3011–3018 (2017). S.C. performed the Engineering Validation Test. L.H., S.C., and L.S. were involved in 30. Akiyama, H. et al. Expression of PDGF-beta receptor, EGF receptor, and receptor planning during the early phase of this project, reviewed the manuscript, and adaptor protein Shc in rat osteoblasts during spaceﬂight. Mol. Cell. Biochem. 202, provided editorial comments. All authors reviewed and contributed to the ﬁnal 63–71 (1999). manuscript. 31. Chakraborty, N. et al. An integrated omics analysis: impact of microgravity on host response to lipopolysaccharide in vitro. BMC Genomics 15, 659 (2014). 32. Barretina, J. et al. CXCR4 and SDF-1 expression in B-cell chronic lymphocytic COMPETING INTERESTS leukemia and stage of the disease. Ann. Hematol. 82, 500–505 (2003). 33. Yonezawa, A., Hori, T., Sakaida, H. & Uchiyama, T. SDF-1 has costimulatory effects The authors declare no competing interests. on human T cells: possible involvement of MAPK (ERK2) activation. Microbiol. Immunol. 44, 135–141 (2000). 34. Meyer-Hoffert, U. et al. Th2- and to a lesser extent Th1-type cytokines upregulate ADDITIONAL INFORMATION the production of both CXC (IL-8 and gro-alpha) and CC (RANTES, eotaxin, Supplementary information is available for this paper at https://doi.org/10.1038/ eotaxin-2, MCP-3 and MCP-4) chemokines in human airway epithelial cells. Int. s41526-020-0106-z. Arch. Allergy Immunol. 131, 264–271 (2003). 35. Pype, J. L. et al. Expression of monocyte chemotactic protein (MCP)-1, MCP-2, and Correspondence and requests for materials should be addressed to A.C.Z. MCP-3 by human airway smooth-muscle cells. Modulation by corticosteroids and T-helper 2 cytokines. Am. J. Respir. Cell. Mol. Biol. 21, 528–536 (1999). Reprints and permission information is available at http://www.nature.com/ 36. Cervantes, J. L. & Hong, B. Y. Dysbiosis and immune dysregulation in outer space. reprints Int. Rev. Immunol. 35,67–82 (2016). 37. Bradley, J. H. et al. Simulated microgravity-mediated reversion of murine lym- Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims phoma immune evasion. Sci. Rep. 9, 14623 (2019). in published maps and institutional afﬁliations. 38. Shi, W. et al. Microgravity induces inhibition of osteoblastic differentiation and mineralization through abrogating primary cilia. Sci. Rep. 7, 1866 (2017). 39. Mitsuhara, T. et al. Simulated microgravity facilitates cell migration and neuro- protection after bone marrow stromal cell transplantation in spinal cord injury. Stem Cell Res. Ther. 4, 35 (2013). Open Access This article is licensed under a Creative Commons 40. Camberos, V. et al. Effects of spaceﬂight and simulated microgravity on YAP1 Attribution 4.0 International License, which permits use, sharing, expression in cardiovascular progenitors: implications for cell-based repair. Int. J. adaptation, distribution and reproduction in any medium or format, as long as you give Mol. Sci. 20, https://doi.org/10.3390/ijms20112742 (2019). appropriate credit to the original author(s) and the source, provide a link to the Creative 41. Baio, J. et al. Cardiovascular progenitor cells cultured aboard the International Commons license, and indicate if changes were made. The images or other third party Space Station exhibit altered developmental and functional properties. npj material in this article are included in the article’s Creative Commons license, unless Microgravity 4, 13 (2018). indicated otherwise in a credit line to the material. If material is not included in the 42. Mehta, S. K. et al. Reactivation of latent viruses is associated with increased article’s Creative Commons license and your intended use is not permitted by statutory plasma cytokines in astronauts. Cytokine 61, 205–209, https://doi.org/10.1016/j. regulation or exceeds the permitted use, you will need to obtain permission directly cyto.2012.09.019 (2013). from the copyright holder. To view a copy of this license, visit http://creativecommons. 43. Sanchez, L. et al. Enrichment of human ESC-derived multipotent mesenchymal org/licenses/by/4.0/. stem cells with immunosuppressive and anti-inﬂammatory properties capable to protect against experimental inﬂammatory bowel disease. Stem Cells 29, 251–262 (2011). © The Author(s) 2020 npj Microgravity (2020) 16 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png npj Microgravity Springer Journals http://www.deepdyve.com/lp/springer-journals/feasibility-potency-and-safety-of-growing-human-mesenchymal-stem-cells-mlnjVKA0GC

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References (50)

S. Cotrupi, Daniela Ranzani, J. Maier (2005)
Impact of modeled microgravity on microvascular endothelial cells.
Biochimica et biophysica acta, 1746 2
C. Thiel, A. Huge, Swantje Hauschild, S. Tauber, B. Lauber, Jennifer Polzer, K. Paulsen, Hartwin Lier, F. Engelmann, B. Schmitz, Andreas Schütte, L. Layer, O. Ullrich (2017)
Stability of gene expression in human T cells in different gravity environments is clustered in chromosomal region 11p15.4
NPJ Microgravity, 3
Y. Tamimi, J. Skarie, T. Footz, F. Berry, B. Link, M. Walter (2006)
FGF19 is a target for FOXC1 regulation in ciliary body-derived cells.
Human molecular genetics, 15 21
V. Camberos, Jonathan Baio, L. Bailey, N. Hasaniya, Larry Lopez, M. Kearns-Jonker (2019)
Effects of Spaceflight and Simulated Microgravity on YAP1 Expression in Cardiovascular Progenitors: Implications for Cell-Based Repair
International Journal of Molecular Sciences, 20
(2000)
possible involvement of MAPK (ERK2) activation
Nanding Wang, Huan Wang, Jun Chen, Xiaofeng Zhang, Juan Xie, Zhi Li, Jing Ma, Wen Wang, Zong-ren Wang (2013)
The simulated microgravity enhances multipotential differentiation capacity of bone marrow mesenchymal stem cells
Cytotechnology, 66
Athena Russell, R. Lefavor, N. Durand, Loren Glover, A. Zubair (2018)
Modifiers of mesenchymal stem cell quantity and quality
Transfusion, 58
Carlos Luna, A. Yew, A. Hsieh (2015)
Effects of angular frequency during clinorotation on mesenchymal stem cell morphology and migration
NPJ Microgravity, 1
M. Zayzafoon, W. Gathings, J. McDonald (2004)
Modeled microgravity inhibits osteogenic differentiation of human mesenchymal stem cells and increases adipogenesis.
Endocrinology, 145 5
Shu-I Pao, Ke-Hung Chien, Hsin‐Ting Lin, Ming‐Cheng Tai, Jiann-Torng Chen, C. Liang (2017)
Effect of microgravity on the mesenchymal stem cell characteristics of limbal fibroblasts
Journal of the Chinese Medical Association, 80
Chaomei Chen, Zhigang Hu, Shengbo Liu, Hung Tseng (2012)
Emerging trends in regenerative medicine: a scientometric analysis in CiteSpace
Expert Opinion on Biological Therapy, 12
J. Pype, L. Dupont, P. Menten, E. Coillie, G. Opdenakker, J. Damme, K. Chung, M. Demedts, G. Verleden (1999)
Expression of monocyte chemotactic protein (MCP)-1, MCP-2, and MCP-3 by human airway smooth-muscle cells. Modulation by corticosteroids and T-helper 2 cytokines.
American journal of respiratory cell and molecular biology, 21 4
J. Barretina, J. Juncà, A. Llano, A. Gutiérrez, A. Flores, J. Blanco, B. Clotet, J. Esté (2003)
CXCR4 and SDF-1 expression in B-cell chronic lymphocytic leukemia and stage of the disease
Annals of Hematology, 82
L. Xue, Yaohui Li, Jun Chen (2017)
Duration of simulated microgravity affects the differentiation of mesenchymal stem cells
Molecular Medicine Reports, 15
Jingsong Yuan, R. Adamski, Junjie Chen (2010)
Focus on histone variant H2AX: To be or not to be
FEBS Letters, 584
E. Rogakou, D. Pilch, A. Orr, V. Ivanova, W. Bonner (1998)
DNA Double-stranded Breaks Induce Histone H2AX Phosphorylation on Serine 139*
The Journal of Biological Chemistry, 273
Jonathan Baio, Aida Martinez, I. Silva, Carla Hoehn, S. Countryman, L. Bailey, N. Hasaniya, M. Pecaut, M. Kearns-Jonker (2018)
Cardiovascular progenitor cells cultured aboard the International Space Station exhibit altered developmental and functional properties
NPJ Microgravity, 4
Cui Zhang, Liang Li, Y. Jiang, Cuicui Wang, Baoming Geng, Yanqiu Wang, Jianling Chen, Fei Liu, Peng Qiu, G. Zhai, Ping Chen, Renfu Quan, Jinfu Wang (2018)
Space microgravity drives transdifferentiation of human bone marrow‐derived mesenchymal stem cells from osteogenesis to adipogenesis
The FASEB Journal, 32
Akihito Yonezawa, T. Hori, H. Sakaida, T. Uchiyama (2000)
SDF‐1 Has Costimulatory Effects on Human T Cells
Microbiology and Immunology, 44
T. Reya, S. Morrison, M. Clarke, I. Weissman (2001)
Stem cells, cancer, and cancer stem cells
Nature, 414
P. Huang, W. Freeman, B. Edenfield, T. Brott, J. Meschia, A. Zubair (2019)
Safety and Efficacy of Intraventricular Delivery of Bone Marrow-Derived Mesenchymal Stem Cells in Hemorrhagic Stroke Model
Scientific Reports, 9
J. Cervantes, B. Hong (2015)
Dysbiosis and Immune Dysregulation in Outer Space
International Reviews of Immunology, 35
E. Karaöz, Pınar Demircan, G. Erman, Eda Güngörürler, Ayla SARIBOYACI (2016)
Comparative Analyses of Immunosuppressive Characteristics of Bone-Marrow, Wharton’s Jelly, and Adipose Tissue-Derived Human Mesenchymal Stem Cells
Turkish Journal of Hematology, 34
F. Pourrajab, S. Forouzannia, S. Tabatabaee (2013)
Molecular characteristics of bone marrow mesenchymal stem cells, source of regenerative medicine.
International journal of cardiology, 163 2
(2018)
How Much Radiation are ISS Astronauts Exposed To
C. Keller, T. Gonwa, D. Hodge, D. Hei, J. Centanni, A. Zubair (2018)
Feasibility, Safety, and Tolerance of Mesenchymal Stem Cell Therapy for Obstructive Chronic Lung Allograft Dysfunction
Stem Cells Translational Medicine, 7
J. Bradley, Shannon Barwick, Gillian Horn, Elizabeth Ullrich, Brianna Best, J. Arnold, R. Gregg (2019)
Simulated microgravity-mediated reversion of murine lymphoma immune evasion
Scientific Reports, 9
Xinjian Mao, Zhe Chen, Qing Luo, Bingyu Zhang, Guanbin Song (2016)
Simulated microgravity inhibits the migration of mesenchymal stem cells by remodeling actin cytoskeleton and increasing cell stiffness
Cytotechnology, 68
(European Space Agency. Studying How Time is Perceived in Space.https://phys.org/news/2019-02-space_1.html (2019).)
European Space Agency. Studying How Time is Perceived in Space.https://phys.org/news/2019-02-space_1.html (2019).
European Space Agency. Studying How Time is Perceived in Space.https://phys.org/news/2019-02-space_1.html (2019)., European Space Agency. Studying How Time is Perceived in Space.https://phys.org/news/2019-02-space_1.html (2019).
Julia Chen, Mardonn Chua, Raymond Bellon, C. Jacobs (2015)
Epigenetic changes during mechanically induced osteogenic lineage commitment.
Journal of biomechanical engineering, 137 2
Bo Yu, Degang Yu, Lei Cao, Xin Zhao, Teng Long, Guangwang Liu, Ting‐ting Tang, Zhen’an Zhu (2011)
Simulated microgravity using a rotary cell culture system promotes chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 MAPK pathway.
Biochemical and biophysical research communications, 414 2
Wen-gui Shi, Yan-fang Xie, Jinpeng He, Jian Zhou, Yu-Hai Gao, Wenjun Wei, Nan Ding, Hui-Ping Ma, C. Xian, Keming Chen, Jufang Wang (2017)
Microgravity induces inhibition of osteoblastic differentiation and mineralization through abrogating primary cilia
Scientific Reports, 7
S. Viswanathan, Yufang Shi, Yufang Shi, J. Galipeau, M. Krampera, K. Leblanc, I. Martin, J. Nolta, D. Phinney, L. Sensébé (2019)
Mesenchymal stem versus stromal cells: International Society for Cellular Therapy Mesenchymal Stromal Cell committee position statement on nomenclature.
Cytotherapy
P. Huang, N. Gebhart, E. Richelson, T. Brott, J. Meschia, A. Zubair (2014)
Mechanism of mesenchymal stem cell-induced neuron recovery and anti-inflammation.
Cytotherapy, 16 10
S. Mehta, B. Crucian, R. Stowe, R. Simpson, C. Ott, C. Sams, D. Pierson (2013)
Reactivation of latent viruses is associated with increased plasma cytokines in astronauts.
Cytokine, 61 1
I. Resnick, C. Barkats, M. Shapira, P. Stepensky, A. Bloom, A. Shimoni, D. Mankuta, N. Varda‐Bloom, L. Rheingold, M. Yeshurun, B. Bielorai, A. Toren, T. Zuckerman, A. Nagler, R. Or (2013)
Treatment of severe steroid resistant acute GVHD with mesenchymal stromal cells (MSC).
American journal of blood research, 3 3
Zhongjun Li, Xingbin Hu, J. Zhong (2019)
Mesenchymal Stem Cells: Characteristics, Function, and Application
Stem Cells International, 2019
T. Mitsuhara, M. Takeda, S. Yamaguchi, Tomotaka Manabe, Masaya Matsumoto, Y. Kawahara, L. Yuge, K. Kurisu (2013)
Simulated microgravity facilitates cell migration and neuroprotection after bone marrow stromal cell transplantation in spinal cord injury
Stem Cell Research & Therapy, 4
N. Chakraborty, Aarti Gautam, S. Muhie, Stacy‐ann Miller, M. Jett, R. Hammamieh (2014)
An integrated omics analysis: impact of microgravity on host response to lipopolysaccharide in vitro
BMC Genomics, 15
(2019)
Studying How Time is Perceived in Space
M. Parra, J. Jung, T. Boone, L. Tran, E. Blaber, Mark Brown, M. Chin, T. Chinn, Jacob Cohen, R. Doebler, Dzung Hoang, Elizabeth Hyde, Matthew Lera, L. Luzod, Mark Mallinson, O. Marcu, Youssef Mohamedaly, A. Ricco, K. Rubins, G. Sgarlato, Rafael Talavera, Peter Tong, E. Uribe, J. Williams, Diana Wu, Rukhsana Yousuf, C. Richey, J. Schonfeld, E. Almeida (2017)
Microgravity validation of a novel system for RNA isolation and multiplex quantitative real time PCR analysis of gene expression on the International Space Station
PLoS ONE, 12
S. Mayer-Wagner, Florian Hammerschmid, J. Redeker, B. Schmitt, B. Holzapfel, V. Jansson, O. Betz, P. Müller (2014)
Simulated microgravity affects chondrogenesis and hypertrophy of human mesenchymal stem cells
International Orthopaedics, 38
L. Sánchez, Iván Gutiérrez-Aranda, G. Ligero, Ruth Rubio, M. Muñoz-Lopez, J. García-Pérez, V. Ramos, P. Real, C. Bueno, R. Rodríguez, M. Delgado, P. Menéndez (2011)
Enrichment of Human ESC‐Derived Multipotent Mesenchymal Stem Cells with Immunosuppressive and Anti‐Inflammatory Properties Capable to Protect Against Experimental Inflammatory Bowel Disease
STEM CELLS, 29
H. Akiyama, Shozo Kanai, M. Hirano, H. Shimokawa, H. Katano, C. Mukai, S. Nagaoka, S. Morita, Y. Kumei (1999)
Expression of PDGF-β receptor, EGF receptor, and receptor adaptor protein Shc in rat osteoblasts during spaceflight
Molecular and Cellular Biochemistry, 202
U. Meyer-Hoffert, D. Lezcano-Meza, J. Bartels, A. Montes-Vizuet, J. Schröder, L. Teran (2003)
Th2- and to a Lesser Extent Th1-Type Cytokines Upregulate the Production of both CXC (IL-8 and Gro-Alpha) and CC (RANTES, Eotaxin, Eotaxin-2, MCP-3 and MCP-4) Chemokines in Human Airway Epithelial Cells
International Archives of Allergy and Immunology, 131
L. Ball, M. Bernardo, H. Roelofs, M. Tol, B. Contoli, J. Zwaginga, M. Avanzini, A. Conforti, A. Bertaina, G. Giorgiani, C. Zijde, M. Zecca, K. Blanc, F. Frassoni, R. Egeler, W. Fibbe, A. Lankester, F. Locatelli (2013)
Multiple infusions of mesenchymal stromal cells induce sustained remission in children with steroid‐refractory, grade III–IV acute graft‐versus‐host disease
British Journal of Haematology, 163
C. Koebnick, A. Langer-Gould, M. Gould, C. Chao, Rajan Iyer, N. Smith, Wansu Chen, S. Jacobsen (2012)
Sociodemographic characteristics of members of a large, integrated health care system: comparison with US Census Bureau data.
The Permanente journal, 16 3
E. Blaber, Natalya Dvorochkin, M. Torres, Rukhsana Yousuf, Brendan Burns, R. Globus, Eduardo Almeida (2014)
Mechanical unloading of bone in microgravity reduces mesenchymal and hematopoietic stem cell-mediated tissue regeneration.
Stem cell research, 13 2
M. Dominici, K. Blanc, Ingo Mueller, I. Slaper-Cortenbach, F. Marini, D. Krause, R. Deans, A. Keating, D. Prockop, E. Horwitz (2006)
Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.
Cytotherapy, 8 4
(Forbes, S. How Much Radiation are ISS Astronauts Exposed To?https://www.forbes.com/sites/quora/2018/11/13/how-much-radiation-are-iss-astronauts-exposed-to/#2949f6ee18a9 (2018).)
Forbes, S. How Much Radiation are ISS Astronauts Exposed To?https://www.forbes.com/sites/quora/2018/11/13/how-much-radiation-are-iss-astronauts-exposed-to/#2949f6ee18a9 (2018).
Forbes, S. How Much Radiation are ISS Astronauts Exposed To?https://www.forbes.com/sites/quora/2018/11/13/how-much-radiation-are-iss-astronauts-exposed-to/#2949f6ee18a9 (2018)., Forbes, S. How Much Radiation are ISS Astronauts Exposed To?https://www.forbes.com/sites/quora/2018/11/13/how-much-radiation-are-iss-astronauts-exposed-to/#2949f6ee18a9 (2018).

Publisher: Springer Journals
Copyright: Copyright © The Author(s) 2020
eISSN: 2373-8065
DOI: 10.1038/s41526-020-0106-z
Publisher site: See Article on Publisher Site

Abstract

www.nature.com/npjmgrav ARTICLE OPEN Feasibility, potency, and safety of growing human mesenchymal stem cells in space for clinical application 1,2 1,2 1,2 1,2 1,2 3 1,2 Peng Huang , Athena L. Russell , Rebecca Lefavor , Nisha C. Durand , Elle James , Larry Harvey , Cuiping Zhang , 4 4 1,2 Stefanie Countryman , Louis Stodieck and Abba C. Zubair Growing stem cells on Earth is very challenging and limited to a few population doublings. The standard two-dimensional (2D) culture environment is an unnatural condition for cell growth. Therefore, culturing stem cells aboard the International Space Station (ISS) under a microgravity environment may provide a more natural three-dimensional environment for stem cell expansion and organ development. In this study, human-derived mesenchymal stem cells (MSCs) grown in space were evaluated to determine their potential use for future clinical applications on Earth and during long-term spaceﬂight. MSCs were ﬂown in Plate Habitats for transportation to the ISS. The MSCs were imaged every 24–48 h and harvested at 7 and 14 days. Conditioned media samples were frozen at −80 °C and cells were either cryopreserved in 5% dimethyl sulfoxide, RNAprotect, or paraformaldehyde. After return to Earth, MSCs were characterized to establish their identity and cell cycle status. In addition, cell proliferation, differentiation, cytokines, and growth factors’ secretion were assessed. To evaluate the risk of malignant transformation, the space-grown MSCs were subjected to chromosomal, DNA damage, and tumorigenicity assays. We found that microgravity had signiﬁcant impact on the MSC capacity to secrete cytokines and growth factors. They appeared to be more potent in terms of immunosuppressive capacity compared to their identical ground control. Chromosomal, DNA damage, and tumorigenicity assays showed no evidence of malignant transformation. Therefore, it is feasible and potentially safe to grow MSCs aboard the ISS for potential future clinical applications. npj Microgravity (2020) 6:16 ; https://doi.org/10.1038/s41526-020-0106-z INTRODUCTION on Earth. On average astronauts on the International Space Station (ISS) received about 10 times more radiation than people on According to the US Census Bureau’s Projections, by the year 2040, Earth . Therefore, aging and radiation-induced organ damage is a 25% of the US population will be 65 years and older . Therefore, the health hazard, especially in long duration spaceﬂight, and is a incidence of age-related diseases such as dementia, neurodegenera- major concern when humans attempt to colonize other planets. tive diseases, stroke, and cancer will continue to increase. Modern Since organ donors will be very scarce in space, the need for medicine is evolving to address the challenges of this rapidly regenerative therapies in this setting will become paramount. increasing public health burden. Currently, the most effective method Mesenchymal stem cells (MSCs) are multipotent cells known to to treat diseased and/or failed organs is organ replacement through modulate immune cell activation and induce tissue repair and transplantation. However, there are not enough donor organs promote regeneration . MSCs are ﬁbroblast-like adherent cells available to meet the needs of the growing number of patients on that primarily differentiate into osteocytes, chondrocytes, and the waiting list. Expansion of stem cells for regenerative medicine 8–10 adipocytes . They secrete a broad range of cytokines and therapies remains a challenge . With the extraordinary growth of growth factors that promote regeneration of other tissue-resident regenerative medicine, the demand for stem cells is greatly stem cells, such as skeletal, hematopoietic, and neural stem cells. increasing. Stem cells in general are quiescent and maintain tight MSCs are identiﬁed in vitro by their plastic adherence and control of their numbers within tissues. It is generally believed that expression of surface markers, such as STRO-1, CD73, CD90, stem cells constitute <1% of cells in any given organ . CD105, CD271, and CD146, as well as lack of expression of mature When induced to proliferate in culture, stem cells can activate hematopoietic lineage markers such as those expressed by B, T, pathways involved in differentiation or senescence and may lose and myeloid cells. They have been used safely in preclinical and their stemness phenotypes after prolonged ex vivo propagation . clinical studies to modulate inﬂammation and induce tissue repair, Most growth media can barely expand stem cells, and if they 11–14 although the mechanisms are not completely understood .We succeed, it tends to be a slow process. Expansion of the large recently showed that interleukin-6 (IL-6) and vascular endothelial numbers of stem cells needed for clinical applications is growth factor play critical roles in MSC-induced neuro-regenera- challenging, and standard culture medium formulations are tion and anti-inﬂammation . The involvement of IL-6 is perplex- limited in their ability to recapitulate physiological growth ing since IL-6 has been well characterized as a pro-inﬂammatory conditions . Since the force of gravity is pervasive and affects cytokine. virtually all aspects of human physiology, we assessed the MSCs are known to respond to applied- or cell-generated feasibility, potency, and safety of expanding stem cells under mechanical forces . MSC’s role in mechano-transduction mechan- true microgravity conditions for human application. isms associated with bone repair and regeneration has been In microgravity, time can appear to pass faster and as a result extensively studied on Earth and to a lesser extent in real and humans in space age slower because of the time-dilation effect . 17–20 Also, exposure to cosmic radiation is much higher in space than simulated microgravity . Unfortunately, it has been 1 2 3 Department of Laboratory Medicine and Pathology, Mayo Clinic, Jacksonville, FL, USA. Center for Regenerative Medicine, Mayo Clinic, Jacksonville, FL, USA. Center for Applied Space Technologies, Merritt Island, FL, USA. BioServe Space Technologies, University of Colorado Boulder, Boulder, CO, USA. email: Zubair.Abba@Mayo.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; P. Huang et al. challenging to compare the data generated from these experi- could be attributed to the freezing technique, which did not ments because of variation in MSC culture methods, the extent of involve the use of controlled rate freezing. To assess sterility, gram population doubling or passaging, use of different growth factors, stain and cultures were performed and found to be negative for tissue source, and donor-to-donor variation. Also, many studies both sMSCs and gMSCs. Following the immunophenotypic criteria utilized MSC cell lines, while others used primary cultures. As such, set forth by the International Society for Cellular Therapy , both the data generated using each of these highly variable sMSCs and gMSCs were stained and analyzed for expression of approaches may not be comparable. CD73, CD90, and CD105, and lack of CD34, CD11b, CD19, HLA-DR, There are conﬂicting reports regarding MSC proliferation and CD45 expression by ﬂow cytometry. Our analysis showed that 21–23 characteristics under simulated microgravity .Thiscan be both sMSCs and gMSCs had comparable expression of triple- attributed to varying experimental approaches, which include positive markers (CD73+, CD90+, CD105+) and absence of different types of microgravity simulators, the use of micro-carriers, mature hematopoietic lineage markers (Fig. 2 and Table 1). levels of oxygenation, and culture media. There are many types of Fluorescence-activated cell sorting sequential gating strategy has microgravity simulators; the most common is a rotating wall vessel. been provided as Supplementary Fig. 1. The rotating wall vessel may not provide true microgravity To assess MSC proliferation potential after space travel, sMSCs conditions, rather it works by altering the direction of gravity with and gMSCs were further cultured to quantify their relative growth respect to the sample over time, or generating rotational centrifugal 18,19 rates. We determined that both sMSCs and gMSCs have forces counteracting the gravitational force . Therefore, space- comparable proliferation rates (Fig. 3). ﬂight experiments like our own are greatly needed to further understand the impacts of real microgravity on MSCs. Despite the limited value of the data generated from simulated Spaceﬂight environment induces changes to cell cycle checkpoint microgravity experiments mainly using animal MSCs, it can be gene expression inferred from these studies that microgravity inhibits the growth Cells preserved in RNAprotect were subjected to RNA isolation and of MSCs by arresting the cells in the G0/G1 phase of the cell cycle real-time PCR for cell cycle checkpoint gene expression analysis (Fig. and diminishes the cellular response to growth factor stimulation. 4). Compared to gMSCs, the expression of CDKN2A,aG1/S Also, microgravity limits MSC’s capacity to undergo osteogenic checkpoint inhibitor, was not signiﬁcantly different in sMSCs after differentiation through regulation of RUNX2 activity and enhances 1 or 2 weeks in culture. This suggests that within 7–14 days culture, adipogenic differentiation by upregulating PPARγ2 activity . microgravity had minimal impact on MSC capacity to enter into the The immediate need for stem cells for regenerative therapies is S phase of cell cycle by altering the expression of CDKN2A.E2F1 on Earth. The Food and Drug Administration released stringent protein is another cell cycle checkpoint inhibitor that preferentially guidelines regarding the use of cellular products for therapeutic purposes outlined under Code of Federal Regulations 1271. These binds to retinoblastoma protein pRB in a cell cycle-dependent standards are universal regardless of the source and method used manner, thereby promoting entry into S phase. Our analysis showed to expand the cells. All cell-based products intended for human that E2F1 expression by sMSCs may be decreased in microgravity use must have established critical quality attributes to ensure environment, although this decrease was not signiﬁcant. Polo-like identity, purity, sterility, and potency of the product. Tumorigenic kinase 1 (PLK1) is a serine/threonine-protein kinase that facilitates potential of the culture-expanded cells must also be evaluated. the transition from G2 to M phase of the cell cycle. PLK1 promotes This is paramount considering the radiation exposure risk in space maturation of the centrosome and establishment of the bipolar is several times higher than the risk on Earth. spindle. Our analysis showed that PLK1 expression by MSCs in In this study, we evaluated the feasibility and safety of growing culture was decreased in microgravity environment, but this MSCs forhuman application atthe ISS. We have established the decrease was only signiﬁcant after 2 weeks in culture on the ISS. identity, purity, viability, and sterility of the space-grown cells In summary, it appears in a 7-day culture, microgravity does not compared to ground controls. We have further assessed the signiﬁcantly alter the expression of CDKN2A or E2F1 or PLK1. functional characteristics of the space-grown cells and assessed their However, with a 14-day culture, microgravity appeared to down- tumorigenic potential. Overall, we have established the feasibility and regulate the expression of PLK1. Therefore, microgravity conditions safety of MSCs grown on the ISS for human application. may slow the progression of MSCs at later stages of the cell cycle during longer-term culture, but this does not appear to reduce their overall growth rate relative to ground controls. Further studies will RESULTS be needed to fully validate this ﬁnding. MSCs maintain their phenotype and proliferative characteristics after expansion on ISS MSCs were seeded in BioCell cassettes (1500 cells/cm ) 3 days Prior microgravity exposure does not affect MSC differentiation before launch and kept in Plate Habitats (PHABs) at 37 °C. Cell capacity imaging was initiated at ISS 2 days after launch. Cells were imaged Osteogenic and adipogenic differentiation assays performed in in real time at multiple time points for 2 weeks and images were our lab after the space-grown cells were returned to Earth beamed down to Earth (Fig. 1). The ground control cells were demonstrated that sMSCs and gMSCs retained similar capacities to imaged simultaneously in our lab. Real-time images of MSCs at ISS differentiate into bone and adipose tissues, as shown with Alizarin showed no obvious differences in morphology and proliferation Red S and Oil Red O stains, respectively (Fig. 5a). To further rate when compared with ground controls. MSCs were cryopre- investigate the effect of microgravity exposure on MSC differ- served in 5% dimethyl sulfoxide and kept frozen at <−95 °C on entiation, we analyzed the expression of genes associated with the ISS until they were returned to Earth using a SpaceX return osteogenic (COL1A1 and RUNX2) and adipogenic (CEBPB) differ- capsule that landed in the Paciﬁc Ocean. Cells were shipped to our entiation by quantitative reverse transcription PCR. Our analysis laboratory at Mayo Clinic Florida within 24 h by FedEx. showed that the expression of the selected genes in space-grown In our lab the space-expanded MSC (sMSCs) were carefully MSCs was statistically similar to ground controls (Fig. 5b). This thawed and we reafﬁrmed their MSC identity, purity, sterility and suggests that MSC expansion for up to 2 weeks in microgravity post-ﬂight proliferation rate compared with the ground control MSCs (gMSCs). Upon thawing, the mean viability of sMSC was environment does not affect their capacity to differentiate into comparable to that of gMSCs (Table 1). This low viability of cells osteoblasts and adipocytes. npj Microgravity (2020) 16 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; P. Huang et al. Fig. 1 Cell culture and real-time imaging. MSCs were seeded and cultured in BioCells cassettes (a) and transported in Plate Habitats (PHABS) (b). Real-time images were taken from ground control and ISS microgravity groups. Imaging started 3 days before launching (L − 3) until 13 days after launching (L + 13) (c). Scale bar: 400 µm. Spaceﬂight environment altered MSC capacity to secrete altered by microgravity environment (Fig. 6). Platelet-derived cytokines and growth factors growth factor-AA subunit (PDGF-AA), a potent growth factor known to induce regeneration and tissue repair, was signiﬁcantly Conditioned media from sMSC and gMSC cultures were evaluated increased and sCD40L, a powerful immunosuppressant, was for cytokine and growth factor secretion proﬁles. Sixty-ﬁve decreased signiﬁcantly after 1 week in microgravity. IL-10, a cytokines and growth factors were evaluated. Secretion of six potent T-helper type 2 (Th2) cytokine with tolerance-inducing cytokines and growth factors were identiﬁed to be signiﬁcantly properties, macrophage chemotactic protein-3 (MCP-3), and Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 16 P. Huang et al. Table 1. Summary of MSC growth characteristics. Pre-ﬂight Post ﬂight Post ﬂight Post ﬂight Post ﬂight Duration in culture (week) 1 1 1 2 2 Gravity Ground Ground ISS Ground ISS Identity % (CD73+, CD90+, CD105+) 89.19 80.2 80.9 88.9 78.6 Viability %, mean ± SD 96 ± 1 60.5 ± 14.9 53.1 ± 12.7 67.4 ± 5.1 54.3 ± 20.2 Mean cell count × 10 per BioCell 0.2–0.5 4.25 ± 2.01 5.12 ± 1.21 5.73 ± 1.80 5.11 ± 2.24 Sterility (Gram stain and culture) Negative Negative Negative Negative Negative Fig. 2 Establishing MSC identity. MSCs thawed from frozen BioCells after 1 week (a–d) and 2 weeks in culture at ISS were tripled stained with CD73, CD90, and CD105 antibodies and then analyzed using ﬂow cytometry. a, c, e, g were from ground control samples. b, d, f, h were from ISS microgravity samples. R7 gate was used to select double positive for CD90 and CD73. R2 gate was applied out of R7 gate to demonstrate triple-positive cells for CD73, CD90, and CD105 (n = 3). stromal cell-derived factor-1α/β (SDF-1α/β), a powerful chemokine evident by less luminescence signal and therefore less amount known to recruit stem cells and immune cells to sites of injury, of ATP in the co-cultured wells. This observation is not due to were signiﬁcantly decreased, while IFN-γ-inducible protein (IP-10) differential proliferation rates of MSCs as two immunomodulation was signiﬁcantly increased after 2 weeks in microgravity environ- potency assays using MSCs from the same donor, with one assay ment relative to ground controls. These observations suggest that involving irradiated non-proliferating MSCs and the other not the effect of microgravity on the secretion of certain cytokines and irradiated (proliferating), yield the same result (see Supplementary growth factors by MSCs is dependent on the duration of exposure Fig. 2). to microgravity conditions. Growing MSCs at the ISS for up to 2 weeks does not compromise Microgravity environment enhanced the immunosuppressive genomic integrity capacity of MSCs To further assess the safety of growing MSCs in space under To evaluate the immunosuppressive capacity of sMSCs compared microgravity conditions, we determined the presence and extent to gMSCs towards peripheral blood mononuclear cells (PBMCs) of DNA damage. This determination was made by visualization in vitro, MSCs were incubated with phytohemagglutinin (PHA)- and subsequent quantiﬁcation of the phosphorylation of histone stimulated PBMCs (Fig. 7). The relative luminescence units (RLUs) variant γH2A.X at serine 139, in the nuclear compartment of cells. from MSCs and PHA-P control wells were subtracted from the RLU Phosphorylation of H2A.X at S139 is one of the initial signaling measurements from experimental wells to correct for ATP events occurring in cells in response to DNA double-strand breaks produced by MSC metabolism. RLU measurements from the wells (DSBs) , and is therefore a well-established marker for DNA containing PBMCs and PHA-P served as a positive control, damage . representative of the maximum PBMC proliferation. This T cell Using immunoﬂuorescence, we could not detect pS.139-γH2A.X proliferation assay was assessed by the amount of ATP content, in the nuclei of cells from any of the experimental conditions which was proportional to the number of metabolically active evaluated; “Ground-1 week” (Fig. 8a1), “Space-1 week” (Fig. 8a7), cells. Rapamycin served as a positive control and could be “Ground-2 weeks” (Fig. 8a13), and “Space-2 weeks” (Fig. 8a19). As observed to suppress the PHA-stimulated T cell proliferation. a positive control, a group of cells from each experimental Compared to gMSCs, sMSCs cultured for 1 or 2 weeks in condition was stimulated with 100 μM etoposide (an inducer of microgravity were signiﬁcantly more immunosuppressive as DNA DSB) for 2 h, and the presence of pS.139-γH2A.X in the npj Microgravity (2020) 16 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA P. Huang et al. Fig. 3 Post-ﬂight MSC proliferation analyses. a MSCs were cultured in triplicates after 1- or 2-week expansion in the ISS environment and on Earth in our laboratory. Cells were seeded into a 96-well plate and stained with IncuCyte Nuclight red to track cell proliferation by nucleus count. Real-time images were acquired by IncuCyte S3 at 2-h interval up to 48 h. Post ﬂight, the ISS-expanded cells proliferated at comparable rate with ground control cells. Statistics determined by one-way ANOVA (n = 3). Error bars represent standard deviation (SD) (n = 3, P > 0.05). b Similarly, assessment of population doubling in longer-term cultures of 1-week ISS-expanded MSCs and the corresponding ground control showed no statistically signiﬁcant difference in growth rate (two-sample Wilcoxon’s rank-sum (Mann–Whitney) test, P = 0.68). Error bars represent standard deviation (SD). nucleus of these cells was readily detectable (Fig. 8a4, 10, 16, 22). stem cells under microgravity conditions for human regenerative For each experimental condition, there was a signiﬁcant difference medicine applications. We chose MSCs because they are the most in the nuclear content of pS.139-γH2A.X between cells treated common type of stem/stromal cell used in clinical trials involving with etoposide and untreated cells (compare Fig. 8a1 with a4, a7 cell therapy. A quick query on clinical trials.gov revealed over 10,000 registered clinical trials involving MSC therapy. Our study is with a10, a13 with a16, and a19 with a22). Similarly as shown in Fig. 8a, quantiﬁcation of pS.139-γH2A.X unique because it involves the use of primary cultures of human MSCs rather than cell lines. Most of the previous reports using ﬂuorescence intensity (Fig. 8b) present in the nuclei of cells MSCs in microgravity conditions were based on human MSC cell showed a signiﬁcant difference between the etoposide-treated lines or cultured animal-derived MSCs. We therefore believe our and -untreated cells in each experimental condition. For all study ﬁndings reﬂect as close to reality in space as is practically conditions assayed, the percentage of pS.139-γH2A.X in the feasible to studying humans under microgravity. untreated cells was <4% when compared to their etoposide- The ﬁrst step towards establishing the clinical utility and safety treated counterparts normalized to 100%. Most importantly, of space-grown cells is ensuring the phenotypic identity of the quantiﬁcation analysis showed that there were no signiﬁcant cells was not changed during growth at ISS under microgravity differences among the four experimental conditions, with very conditions. Our evaluation showed that MSCs maintained their similar percentages of pS.139-γH2A.X reported for each one: physical identity and proliferative characteristics aboard ISS. In “Ground-1 week”—3.7%, “Space-1 week”—3.4%, “Ground- addition, these physical attributes were maintained after they 2 weeks”—3.3%, and “Space-2 weeks”—3.1% (Fig. 8b). were returned to Earth and further expanded under standard These data demonstrate that the MSC nuclear content of gravity conditions in our lab. We could not ﬁnd previous studies pS.139-γH2A.X, and therefore associated DNA DSBs, is not affected that evaluated MSC surface marker identity in real microgravity. by growth in microgravity conditions for up to 2 weeks. However, there were many studies that assessed MSC morpho- In addition, chromosomal studies were performed using logical characteristics under simulated microgravity. Luna et al. conﬂuent cultures of sMSCs and gMSCs. Karyotyping using reported that MSCs responded to clinorotation by adopting a standard G banding method showed no chromosomal abnorm- more rounded, less-spread morphology. alities (Fig. 9). It was challenging to accurately evaluate MSC proliferation rate in real time while in culture at ISS. Resources at ISS and crew time MSCs showed no evidence of tumorigenic transformation after a were the main limiting factors. We resorted to a series of simple 14-day culture aboard ISS microscopic images. These images were sent to us in near real Although we did not detect any evidence of compromised time. In addition, based on a prior experience with ﬁbroblast cell genomic integrity, there is still concern of tumorigenic transfor- lines, our MSCs had to be seeded on Earth to enable the cells to be mation due to the presence of cosmic radiation that may result in adherent, as gravity is apparently needed for this process. Seeding oncogenic mutation. We therefore performed a tumorigenicity the MSCs at ISS under microgravity may have led to non- assay to evaluate both sMSC and gMSC tumorigenic potential at 1- adherence and subsequent cell death. We opted to pre-seed the and 2-week cultures. HT-1080 was used as a positive control cell MSCs on Earth. Post-ﬂight MSC count and viability were impacted line (Fig. 10a) and W138 as negative control cell line (Fig. 10b). by cryopreservation method. Overall, our assessment was that the Neither sMSCs nor gMSCs showed evidence of tumor formation cell counts between ISS-expanded and ground control cells were after a 1- or 2-week culture. comparable. Therefore, it is unclear if there is any difference in proliferation rate between the ISS and ground control cells. However, analysis of cell cycle checkpoint gene expression DISCUSSION showed that the spaceﬂight environment after a 14-day culture The focus of this study was to evaluate the feasibility and safety of caused downregulation of PLK1, a late G1/S and G2/M checkpoint using space-grown MSCs for potential future clinical application promoter. This suggests that microgravity could slow MSC on Earth and during long-term spaceﬂight. Many studies have proliferation in longer-term cultures. Further studies will be shown detrimental effects of the absence of gravity on bone needed to fully elucidate this observation. homeostasis and the musculoskeletal system. In this study, we Our studies showed that prior microgravity exposure does not aimed to identify the beneﬁcial effects of microgravity on stem affect subsequent MSC differentiation on Earth. However, previous cell proliferative capacity with the long-term objective of growing studies of MSC grown in microgravity demonstrated a skewing of Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 16 P. Huang et al. Fig. 4 Impact of microgravity on expression of cell cycle checkpoints associated genes. MSCs were culture for 1 and 2 weeks. Cells were harvested and saved in RNAprotect. RNA was later isolated and quantitative reverse transcription PCR (RT-qPCR) analysis was performed as described in the “Methods” section. Expression of cell cycle checkpoint-associated genes: a cyclin-dependent kinase inhibitor 2A (CDKN2A), b transcription factor (E2F1), and c serine/threonine-protein kinase, known as polo-like kinase 1 (PLK1) were evaluated and compared to ground control gMSCs. Gene expression was normalized to GAPDH. Statistics determined by one-way ANOVA (n = 3, *P < 0.05), error bars represent standard deviation (SD). 24,29 their differentiation capacity towards adipogenic lineage . Our that CXCR4, a receptor for SDF-1, is overexpressed in simulated results do not support these reported studies. Possible explana- microgravity. Similarly, Dr. Kearns-Jonker’s group reported that tion as to why our study differs from these reports could be that simulated microgravity induces cardiac progenitor cells (CPCs) to the effects of microgravity on MSC differentiation to adipogenic increase SDF-1a messenger RNA (mRNA) expression and this effect lineage require longer microgravity exposure beyond 2 weeks was more pronounced in adult CPCs . They also used BioCell culture. Further studies will be needed to determine if prolonged Culture Cassette to grow CPCs at ISS and observed similar SDF-1a exposure to microgravity beyond 2 weeks in culture has lasting mRNA overexpression . Although these ﬁndings were based on effects on the MSC differentiation capacity. mRNA transcripts in CPC, while ours measured protein secretion Our studies showed that the spaceﬂight environment altered by bone marrow-derived MSCs, our study appears to contradict MSC capacity to secrete certain cytokines and growth factors and the observation by Dr. Kearns-Jonker’s group. This differential this effect was more enhanced with time. MSC capacity to secrete ﬁnding highlights the fact that the effect of microgravity varies PDGF-AA and IP-10 was signiﬁcantly increased, while MCP-3 signiﬁcantly among different cell types as well as cells at different (CCL7), IL-10, sCD40L, and SDF-1 (CCL12) was signiﬁcantly stages of their life cycles. Also, mRNA expression may not decreased in microgravity environment relative to ground necessarily correlate with protein expression and secretion. More controls. Contrary to our ﬁndings, it was previously reported that studies will be needed to further investigate these observations. microgravity reduces the expression of PDGF-β receptor in rat Our studies showed that microgravity environment enhanced osteoblasts . Another study reported that human endothelial the immunosuppressive capacity of MSCs. It is established that cells in response to lipopolysaccharide stimulation under micro- microgravity environment and space radiation signiﬁcantly affect 31 15 gravity increased the expression of PDGF . MCP-3 and SDF-1 are the immune system . Immune dysregulation in microgravity both potent chemotactic factors known to play signiﬁcant roles in conditions is not yet fully understood. It is known that cytotoxic T immune regulation, bone formation, and recruitment of cell function is diminished and this is correlated with reactivation 42 42 T lymphocytes and monocytes to local inﬂammatory of latent herpesviruses in some crewmembers . Mehta et al. 32–35 responses . Both immunity and bone formation are known showed a direct correlation of plasma cytokine alterations with 36–38 to be altered in microgravity . It has been suggested that MCP viral shedding in speciﬁc crewmembers, and hypothesized that expression is affected by simulated microgravity. Our study spaceﬂight is associated with Th2-type tolerogenic immune addresses the impact of true microgravity on secretion of MCP- responses . On the contrary, both sCD40L, a powerful immuno- 3. Using rat bone marrow-derived MSC, Mitsuhara et al. reported suppressant, and IL-10, a Th2-type cytokine, secretions by sMSCs npj Microgravity (2020) 16 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA P. Huang et al. Fig. 5 Inﬂuence of microgravity on osteogenic and adipogenic differentiation. sMSC and gMSC cultures were stained with Alizarin Red and Oil Red O (a). Each condition was seeded and measure in triplicates. Representative pictures (×100) are shown from post-ﬂight MSC culture in a growth medium or differentiation medium after 2-week culture expansion at ISS or ground (Earth). Quantitative real-time PCR analysis of osteogenic (COL1A1, RunX2) and adipogenic (CEBPB) genes was done after 21 days in differentiation medium (b). RNA from three 1-week and 2-week MSC cultures were used for quantitative PCR analysis. Gene expression was normalized to GAPDH. Statistics determined by one-way ANOVA (n= 3). Error bars represent standard deviation (SD). Scale bar: 100 µm. were signiﬁcantly decreased in our study. In agreement with these and many limitations. Access to ISS is very limited. During the reports, our T cell proliferation assay showed sMSCs to have planning and execution of our study, only SpaceX in the United enhanced immunosuppressive properties via a mechanism that is States could provide the required up mass to ISS as well as the unlikely to be dependent on IL-10 and sCD40L. Our study desired down mass following the conduct of our research. Other opportunities to access ISS and return payloads to Earth are highlights a potential role of MSCs in the mechanism of spaceﬂight-induced immune dysregulation and tolerance. How- currently in development and might soon become available. In ever, further studies will be needed in order to fully understand addition, opportunities for suborbital studies have since become the mechanism of microgravity-induced immunosuppression. available, such as Blue Origin and the Northrup Grumman Cygnus On the ISS, astronauts receive more than ten times the amount capsule now regularly launches to the ISS. We were very limited by of space and cosmic radiation compared to what humans receive the weight and volume of our payload. We had to modify our plan on Earth. This is despite the fact that the ISS orbit does not travel to eliminate media exchanges and minimize the amounts of through the Van Allen radiation belts. In contrast, long duration reagents and supplies to be used due to reduced crew time and spaceﬂight outside the Van Allen radiation belts could be very cold/warm stowage space on ascent to and descent from the ISS. hazardous to humans. This could result in signiﬁcant DNA damage Use of only one donor in our studies is not ideal as we have and genomic instability that may lead to cancer. This is particularly previously reported that MSC characteristics vary signiﬁcantly concerning if stem cells are affected. Our studies showed growing from donor to donor . Crew time is signiﬁcantly limited as they MSCs at ISS for 2 weeks does not compromise genomic integrity are tasked with multiple projects. Automated bioreactors that are or evidence of tumorigenic transformation. now offered by multiple implementation partners are very useful Even though we have successfully demonstrated that MSCs can and can mitigate some of the challenges highlighted, such as crew be safely expanded at ISS, our study faced signiﬁcant challenges time expenditure and complexity of operating equipment at ISS. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 16 P. Huang et al. Fig. 6 Conditioned media from sMSC and gMSC cultures were evaluated for cytokine and growth factor secretion proﬁle. PDGF-AA (a) was increased and sCD40L (i) was decreased signiﬁcantly after 1 week in microgravity. IL-10 (d), MCP-3 (f), and SDF-1α/β (h) were decreased and IP-10 (l) was increased signiﬁcantly in microgravity environment after 2 weeks in microgravity environment relative to ground controls. Statistics determined by Student’s t test (n = 3, *P < 0.05 **P < 0.01). Error bars represent standard deviation (SD). In summary, it is feasible and safe to grow MSCs aboard the ISS. Space-grown MSCs from our study maintained their morphologi- cal and phenotypic characteristics, proliferative capabilities, differentiation potential, and were even more potent in their immunosuppressive abilities compared to ground control MSCs. These ﬁndings are positive indications that MSCs grown in microgravity could be used for future clinical applications. Our studies demonstrate the feasibility and safety of expanding MSCs at ISS for possible clinical application, but more studies are needed to fully validate our ﬁndings. METHODS Experimental design The experiments utilized BioServe’s single-well BioCell, a cell culture spaceﬂight certiﬁed piece of hardware that can effectively support cell culture. The experiment utilized 17 BioCells containing MSCs were ﬂown in PHABs for transportation to ISS. Once on board the ISS, the PHABs were placed inside of BioServe’s SABL unit, which provides 37 °C temperature Fig. 7 Immunomodulation potency assay comparing the immu- control and 5% CO . The experiment also utilized BioServe’s standard nosuppressant capacity of gMSC and sMSC towards peripheral inverted phase contrast microscope for imaging of the cells once on board blood mononuclear cell (PBMC). MSCs were incubated for 72 h with ISS. It also required samples be frozen at <−95 °C once processed and phytohemagglutinin (PHA) stimulated PBMCs at 1:3 ratio of MSCs to remain frozen until return to Earth. MSC cultures were processed at two PBMCs. Luminescence is proportional to the amount of ATP present, time points: at 7 and 14 days. One BioCell was ﬂown speciﬁcally for and the amount of ATP is proportional to the number of imaging, it was imaged every 48 h for 14 days. MSCs were imaged at ×40 metabolically active cells present. Luminescence fold change is and ×100 magniﬁcations using the VUE camera system from within the relative to the positive control (PBMCs with PHA). Each condition microgravity science glovebox. The microscope utilized at the ISS was a was seeded and measured in quintuplicate. Statistics determined by standard phase contrast inverted microscope (Nikon TS100). A high- one-way ANOVA (n = 5, *P < 0.05, **P < 0.005). Error bars represent resolution camera attached to the microscope was able to capture both standard deviation (SD). still and video images once the cultures were removed from the on-orbit incubator and placed on the microscope stage. Live pictures were taken However, they are very expensive and may consume a signiﬁcant for each imaging BioCell periodically and sent to Earth in real time. At the part of the research budget. We believe the cost will come down end of the experiment, 5 ml 20% paraformaldehyde (PFA; Electron once the commercial crew vehicles come online. This will Microscopy Sciences) (ﬁnal concentration is 4%) was added to the imaging signiﬁcantly increase available crew time on board the ISS to BioCells directly to preserve the cells at <−95 °C until return to Earth. complete scientiﬁc studies such as ours. Our spaceﬂight studies Similarly, gMSCs were treated exactly the same as those sMSCs in answered many of our original questions; however, we are now microgravity environment. Even the timing of cell evaluation and cell left with more questions than answers and the desire to return to preservation was approximately maintained. Results of experiments shown ISS to perform more experiments. in Figs. 3 and 5 involved post-ﬂight Earth re-expanded cells. npj Microgravity (2020) 16 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA P. Huang et al. Fig. 8 Genomic integrity analysis of the safety of growing MSC in space. a Bone marrow-derived mesenchymal stem cells (5000 cells/ well) previously grown for 1 week or 2 weeks in space, along with ground control cells were seeded to ibidi µ-Slide 8 Well. Cells were stimulated with 100 μM etoposide as indicated, ﬁxed, and subjected to immunoﬂuorescence analysis to visualize pS139-H2A.X. Scale bars indicate 10 μm. b shows quantitation of the ﬂuorescence intensity of pS139-H2A.X (n = 40 cells). Analysis was conducted using the Image J software. Fluorescence intensities were normalized to the etoposide-treated group for each experimental condition. **** indicates statistical signiﬁcance (<0.0001) determined by one-way ANOVA as compared to cells treated with 100 μM etoposide. Error bars represent standard deviation (SD). fetal bovine serum (FBS; R&D Systems) (designated as Complete Culture 20,21 Medium, CCM) according to a previously reported method . MSCs were expanded to passage 2 and MSC cultures were over 89% pure based on the CD73, CD90, and CD105 co-expression and absence of hematopoietic lineage-speciﬁc markers. Expanded MSCs in 25 ml of CCM were seeded into BioCells at passage 4. BioCells were divided into 1- or 2-week groups. The 1-week group was seeded with 5 × 10 MSCs, while the 2-week group was seeded with 2 × 10 MSCs. Identical BioCells were prepared for ground controls. After launch, no media exchange was performed. At the speciﬁed time points, MSCs were treated and harvested according to the following procedures. A 4.5 ml media sample was pulled into a monovette through a BioCell sample port and frozen at −95 °C directly for cytokine and growth factor analysis. Cells in the BioCell were trypsinized and 1.5 ml of cell sample was mixed with 5 ml of RNAprotect (Qiagen), and then moved to −95 °C for RNA expression analysis. The remaining cells were frozen in cell preserving medium containing 5% dimethyl sulfoxide (Sigma-Aldrich) directly inside the BioCells and stored at −95 °C. Viability post thawing: Immediately after the BioCells were thawed, aliquots were made for further experiments. Cell number and viability per BioCell were evaluated via Trypan blue (0.4%; VWR) and documented. Post-ﬂight real-time proliferation analysis: Six sMSC and gMSC aliquots from 12 BioCells were selected for real-time cell proliferation analysis post ﬂight. Each aliquot was expanded and 2 × 10 cells/well seeded into a 96- well plate. IncuCyte NucLight Red ﬂorescence dye (Essen BioScience) was then added to each well at 1:500 dilution. The 96-well plate-containing cells was loaded into the IncuCyte (Essen Bioscience) with scheduled scanning at 2-h intervals up to 48 h. Automated cells counts from each well were further analyzed using the Prism 8 software (GraphPad). MSC phenotyping by ﬂow cytometry Expanded MSCs were harvested with 0.05% Trypsin (Fisher Scientiﬁc). Cells were pelleted at 300 × g for 5 min, re-suspended in 2 ml of phosphate- buffered saline (PBS) and ﬁltered through a 35-µm nylon-mesh ﬁlter (Fisher Scientiﬁc), pelleted again at 300 × g for 5 min, and re-suspended to a ﬁnal volume of 1 ml of PBS. Filtered cells were dispensed to 100 μl aliquots, incubated with MSC marker antibodies against CD73, CD90, and CD105 (BD Biosciences, Human MSC Analysis Kit, Catalog number 562245, Lot number 5313719, 1:100 diluted in BD Pharmingen™ stain buffer), plus antibodies against negative markers CD34, CD11b, CD19, HLA-DR, and CD45 . Cells were incubated with antibodies in the dark for 20 min at room temperature. Stained cells were resuspended in a volume of 600 μl with the addition of 500 μl of PBS prior to analysis by ﬂow cytometry. Cell cycle analysis RNA was extracted using RNeasy Plus Mini Kit (Qiagen) as was previously reported and was prepared for real-time PCR assays. Three biological samples were selected for each group. TaqMan Gene Expression Assays were purchased from Applied Biosystems: GAPDH (Hs02758991_g1), CDKN2A (Hs00923894_m1), E2F1 (Hs00153451_m1), and PLK1 (Hs00153444_m1). TaqMan Fast Advanced Master Mix (Applied Biosystem) Cell culture and proliferation were used according to the manufacturer’s user guide. Each sample was −ΔΔCT Pre-ﬂight mononuclear cells were isolated from a healthy bone marrow tested in triplicate. GAPDH was used as the internal control and the 2 donor using Histopaque-1077 (Sigma-Aldrich) following density gradient method was used to analyze data. GAPDH is a stable housekeeping gene 45–47 protocols and used to culture expand the MSCs. The MSCs were cultured in both on Earth and under microgravity condition . For statistical α-minimum essential medium (Fisher Scientiﬁc) supplemented with 16.5% analysis, unpaired T tests were carried out using GraphPad Prism 8. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 16 P. Huang et al. Fig. 9 Chromosomal studies were performed using conﬂuent cultures of sMSCs and gMSCs. Twenty cells were analyzed from sMSCs (a) and gMSCs (b) and two of these were karyotyped using standard G banding method. No chromosomal abnormalities were detected. Osteogenic and adipogenic differentiation assays in quintuplicate at 6.6 × 10 cells/well and stored in a humidiﬁed 37 °C, 5% CO incubator for 2 h. Following incubation, fresh, primary PBMCs (Astarte Bone differentiation media (BDM) contained CCM plus 1 nM dexametha- 2 Biologics) were re-suspended in a medium (RPMI 1650 medium, 5% FBS, sone (Sigma-Aldrich), 20 mM β-glycerolphosphate (Sigma-Aldrich), and 1× Glutamax, 0.05% penicillin/streptomycin; Fisher Scientiﬁc) and seeded 50 µg/ml L-ascorbic acid 2-phosphate (Sigma-Aldrich) at a ﬁnal concentra- to wells containing MSCs at 2 × 10 cells/well, resulting in a 1:3 ratio of tion. Fat differentiation media (FDM) contained CCM plus 0.5 µM MSCs to PBMCs. PHA-P (Sigma-Aldrich), a mitogen that activates dexamethasone, 0.5 µM isobutylmethylxanthine (Sigma-Aldrich) and proliferation of T cells, was added to all wells containing MSCs at a 50 µM indomethacin (Sigma-Aldrich) at ﬁnal concentration. MSCs were concentration of 10 µg/ml. Additional control conditions were included, seeded at 1 × 10 cells/well in 6-well plates. Cultures were propagated in which contained PBMCs only, PBMCs with PHA-P, and PBMCs with PHA-P CCM until MSC reached 100% conﬂuence. CCM was then replaced with and 10 µg/ml rapamycin (InvivoGen), an IL-2 inhibitor to serve as a BDM and FDM for induction of bone differentiation and fat differentiation. functional control of immunosuppression. The plates were incubated in a Cells were washed with PBS and differentiation media replaced every humidiﬁed 37 °C, 5% CO incubator for 72 h. Following 72 h of incubation, 3 days for 21 days. After 21 days, media were aspirated and cells were the plates were removed and the quantity of ATP in each well was washed with PBS. Two milliliters of 10% formalin (Fisher Scientiﬁc) was measured via luminescence using CellTiter-Glo Luminescent Cell Viability added to each well and cells were incubated for 1 h at room temperature. Kit (Promega), which implements a thermostable luciferase that produces Formalin was aspirated and bone differentiation wells were washed with luminescent signal proportional to the amount of ATP present in the deionized (DI) water, while fat differentiation wells were washed with PBS. sample. The RLUs from MSC and PHA-P control wells were subtracted from One percent Alizarin Red S (Sigma-Aldrich) in DI water was used to stain the RLU measurements from experimental wells to correct for ATP cells for osteogenesis. For adipogenesis staining, ﬁrst 0.5% Oil Red O produced by MSC metabolism. RLU measurements from the wells (Sigma-Aldrich) in isopropyl alcohol was diluted with PBS to make a 0.3% containing PBMCs and PHA-P served as a positive control, representative working solution. Cells were then stained for adipogenesis with 0.3% Oil of the maximum PBMC proliferation. The normalized data was presented Red O. After 20 min at room temperature, the solution was aspirated and as a fold change in comparison to the positive control condition (PBMCs cells were washed with DI water and PBS until wash ﬂuid became clear. with PHA-P). Statistical signiﬁcance was determined by one-way analysis of Each condition was seeded and measure in quintuplicates. Stained cells variance, comparing the means of each condition. were then imaged using Olympus inverted microscope at ×100 magniﬁcation. Chromosomal analysis Chromosomal karyotype was prepared from adherent MSC in a T25 ﬂask Quantitative real-time PCR grown to conﬂuence. At this point, 25 µl of 1 µg/ml colcemid solution RNA from MSCs that has been induced under osteogenic and adipogenic (Sigma-Aldrich) per 2 ml of media was added to the ﬂask and cells were differentiation was extracted using RNeasy Plus Mini Kit as was previously incubated at 37 °C with 5% CO ,5% O , and 90% N for 16–22 h. The 2 2 2 reported and then prepared for real-time PCR assays. Quantitative real- medium was removed from the ﬂask and placed in a 15 ml centrifuge tube time PCR analysis of osteogenic (COL1A1, Hs00164004_m1; RunX2, and TrypLE Express was used to dissociate the cells. Harvested cells were Hs00231692_m1) and adipogenic (CEBPB, Hs00270923_s1) genes was added to the 15 ml centrifuge tube and centrifuged at 300 × g for 8 min. done after MSCs have been exposed in a differentiation medium for The medium was removed and the pellet was re-suspended in 10 ml of 21 days. 50:50 hypotonic solution (potassium chloride 0.075 M:sodium citrate 0.8%) and incubated at RT for 20–30 min. Two milliliters of 3:1 ﬁxative (methanol: Cytokines and growth factor analysis glacial acetic acid) was added and cells were centrifuged. Pelleted cells Cell culture media collected directly from BioCells on the ISS were thawed were re-suspended in 10 ml of 3:1 ﬁxative (methanol:glacial acetic acid). and aliquoted after we received the payload on Earth. Conditioned Centrifugation and addition of ﬁxative was repeated two more times. Cells medium from three BioCells per time point (1 or 2 weeks in culture) were were dropped on pretreated microscope slides at 25 °C and 65% relative aliquoted into 200 µl samples. Secreted cytokines and growth factors were humidity. Twenty cells were analyzed and two of these were karyotyped analyzed using the Human Cytokine/Chemokine 65-Plex Panel (HD65) (Eve using standard G banding method. Technologies). Each sample was analyzed in duplicate, and averaged to represent the cytokine/chemokine value. Student’s t test was used to DNA damage assay compare signiﬁcance between ground and microgravity (ISS) samples. Bone marrow-derived MSCs (5 × 10 cells/well) were seeded to µ-Slide 8 Details cytokines/chemokines list from Eve Technologies has been Well (ibidi) grown to ~70% conﬂuency and stimulated with 100 μM included in Supplementary Fig. 3. etoposide (Abcam) for 2 h as indicated. Following etoposide treatment, cells were washed three times with PBS and ﬁxed with 4% PFA (37 °C, Immunosuppression assay 15 min). After ﬁxation, cells were washed three times with PBS, Cryopreserved MSCs were thawed in a 37 °C water bath and re-suspended permeabilized with 0.1% Triton-X 100 in PBS for 4 min (RT), and blocked in CCM. In white 96-well plates (Greiner, Kremsmünster) MSCs were seeded with 3% bovine serum albumin and 0.05% Tween-20 in PBS (blocking npj Microgravity (2020) 16 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA P. Huang et al. Fig. 10 Tumorigenicity assay. Cells were seeded on a soft agar to evaluate their tumorigenic potential. Five thousand cells/well of positive control cell line HT-1080 (a) and 67,000 cells/well of negative control cell line W138 (b) were used as control to validate the procedure. Twelve cells/well of MSCs were used for this assay. (c) Ground control and (d) microgravity were cells that were culture expanded for 1 week and (e) ground control and (f) microgravity were cells cultured for 2 weeks on Earth and in microgravity, respectively. solution) for 1 h at RT. Samples were incubated with pS.139-H2A.X 4. Russell, A. L., Lefavor, R., Durand, N., Glover, L. & Zubair, A. C. Modiﬁers of antibody (Cell Signaling Technology) in blocking solution at 1:250 for mesenchymal stem cell quantity and quality. Transfusion 58, 1434–1440 (2018). 24 h at 4 °C, washed ﬁve times with PBS and further incubated with goat 5. European Space Agency. Studying How Time is Perceived in Space. https://phys. anti-rabbit immunoglobulin G (H + L) secondary antibody (Fisher org/news/2019-02-space_1.html (2019). Scientiﬁc) at 1:800 and 4′,6-diamidino-2-phenylindole (Sigma-Aldrich) at 6. Forbes, S. How Much Radiation are ISS Astronauts Exposed To? https://www.forbes. 1:5000 in blocking solution for 2 h at RT. Subsequently, cells were washed com/sites/quora/2018/11/13/how-much-radiation-are-iss-astronauts-exposed-to/ ﬁve times with PBS, mounting media (ibidi) was added to the wells, and #2949f6ee18a9 (2018). images were captured using the EVOS FL Cell Imaging System (Fisher 7. Viswanathan, S. et al. Mesenchymal stem versus stromal cells: International Scientiﬁc). Society for Cell & Gene Therapy (ISCT(R)) Mesenchymal Stromal Cell committee position statement on nomenclature. Cytotherapy 21, 1019–1024 (2019). 8. Li, Z., Hu, X. & Zhong, J. F. Mesenchymal stem cells: characteristics, function, Tumorigenicity assay and application. Stem Cells Int. 2019, https://doi.org/10.1155/2019/8106818 Soft agar assay was used to evaluate tumorigenicity of cells grown in (2019). microgravity as well as ground controls. Human ﬁbrosarcoma cell line HT- 9. Karaoz, E., Cetinalp Demircan, P., Erman, G., Gungorurler, E. & Eker Sariboyaci, A. 1080 (ATCC, CCL-121) and ﬁbroblast cell line WI38 (ATCC, CCL-75) were Comparative analyses of immunosuppressive characteristics of bone-marrow, used as positive and negative controls. Cells were suspended in 2 ml of top Wharton’s jelly, and adipose tissue-derived human mesenchymal stem cells. Turk. agar (0.35% agar in α-minimum essential medium containing 20% FBS) J. Haematol. 34, 213–225 (2017). and then plated on 3 ml of bottom agar (0.5% agar in α-minimum essential 10. Pourrajab, F., Forouzannia, S. K. & Tabatabaee, S. A. Molecular characteristics of medium containing 20% FBS) in a 6-well plate. Colonies were counted after bone marrow mesenchymal stem cells, source of regenerative medicine. Int. J. a 14-day incubation. Cardiol. 163, 125–131 (2013). 11. Keller, C. A. et al. Feasibility, safety, and tolerance of mesenchymal stem cell therapy for obstructive chronic lung allograft dysfunction. Stem Cells Transl. Med. Reporting summary 7, 161–167 (2018). Further information on research design is available in the Nature Research 12. Huang, P. et al. Safety and efﬁcacy of intraventricular delivery of bone marrow- Reporting Summary linked to this article. derived mesenchymal stem cells in Hemorrhagic Stroke Model. Sci. Rep. 9, 5674 (2019). 13. Resnick, I. B. et al. Treatment of severe steroid resistant acute GVHD with DATA AVAILABILITY mesenchymal stromal cells (MSC). Am. J. Blood Res. 3, 225–238 (2013). The authors declare that the data that support the ﬁndings of this study are available 14. Ball, L. M. et al. Multiple infusions of mesenchymal stromal cells induce sustained within the paper. remission in children with steroid-refractory, grade III-IV acute graft-versus-host disease. Br. J. Haematol. 163, 501–509 (2013). 15. Huang, P. et al. Mechanism of mesenchymal stem cell-induced neuron recovery Received: 9 December 2019; Accepted: 7 May 2020; and anti-inﬂammation. Cytotherapy. https://doi.org/10.1016/j.jcyt.2014.05.007 (2014). 16. Chen, J. C., Chua, M., Bellon, R. B. & Jacobs, C. R. Epigenetic changes during mechanically induced osteogenic lineage commitment. J. Biomech. Eng. 137, 020902 (2015). REFERENCES 17. Pao, S. I. et al. Effect of microgravity on the mesenchymal stem cell characteristics 1. Koebnick, C. et al. Sociodemographic characteristics of members of a large, of limbal ﬁbroblasts. J. Chin. Med. Assoc. 80, 595–607 (2017). integrated health care system: comparison with US Census Bureau data. Perm J. 18. Blaber, E. A. et al. Mechanical unloading of bone in microgravity reduces 16,37–41 (2012). mesenchymal and hematopoietic stem cell-mediated tissue regeneration. Stem 2. Chen, C., Hu, Z., Liu, S. & Tseng, H. Emerging trends in regenerative medicine: a Cell Res. 13, 181–201 (2014). scientometric analysis in CiteSpace. Expert Opin. Biol. Ther. 12, 593–608 (2012). 19. Mao, X., Chen, Z., Luo, Q., Zhang, B. & Song, G. Simulated microgravity inhibits the 3. Reya, T., Morrison, S. J., Clarke, M. F. & Weissman, I. L. Stem cells, cancer, and migration of mesenchymal stem cells by remodeling actin cytoskeleton and cancer stem cells. Nature 414, 105–111 (2001). increasing cell stiffness. Cytotechnology 68, 2235–2243 (2016). Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2020) 16 P. Huang et al. 20. Yu, B. et al. Simulated microgravity using a rotary cell culture system promotes 44. Tamimi, Y. et al. FGF19 is a target for FOXC1 regulation in ciliary body-derived chondrogenesis of human adipose-derived mesenchymal stem cells via the p38 cells. Hum. Mol. Genet. 15, 3229–3240 (2006). MAPK pathway. Biochem. Biophys. Res. Commun. 414, 412–418 (2011). 45. Cotrupi, S., Ranzani, D. & Maier, J. A. Impact of modeled microgravity on micro- 21. Wang, N. et al. The simulated microgravity enhances multipotential differentia- vascular endothelial cells. Biochim. Biophys. Acta 1746, 163–168 (2005). tion capacity of bone marrow mesenchymal stem cells. Cytotechnology 66, 46. Thiel, C. S. et al. Stability of gene expression in human T cells in different gravity 119–131 (2014). environments is clustered in chromosomal region 11p15.4. npj Microgravity 3,22 22. Zayzafoon, M., Gathings, W. E. & McDonald, J. M. Modeled microgravity inhibits (2017). osteogenic differentiation of human mesenchymal stem cells and increases 47. Parra, M. et al. Microgravity validation of a novel system for RNA isolation and adipogenesis. Endocrinology 145, 2421–2432 (2004). multiplex quantitative real time PCR analysis of gene expression on the Inter- 23. Mayer-Wagner, S. et al. Simulated microgravity affects chondrogenesis and national Space Station. PLoS ONE 12, e0183480 (2017). hypertrophy of human mesenchymal stem cells. Int. Orthop. 38, 2615–2621 (2014). 24. Zhang, C. et al. Space microgravity drives transdifferentiation of human bone ACKNOWLEDGEMENTS marrow-derived mesenchymal stem cells from osteogenesis to adipogenesis. We acknowledge and thank the Center for the Advancement of Science in Space FASEB J. 32, 4444–4458 (2018). (CASIS), now renamed as National Laboratory, Mayo Clinic Center for Regenerative 25. Dominici, M. et al. Minimal criteria for deﬁning multipotent mesenchymal stromal Medicine, BioServe, and the following members of CAST (Center for the Applied cells. The International Society for Cellular Therapy position statement. Cyto- Space Technology) for their support during the early phase of this project: Maria therapy 8, 315–317 (2006). Peterson, Bob Twiggs, and Richard Snyder, Director, University of Florida Center of 26. Rogakou, E. P., Pilch, D. R., Orr, A. H., Ivanova, V. S. & Bonner, W. M. DNA double- Excellence for Regenerative Health Biotechnology. This project was funded by Center stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. for the Advancement of Science in Space (Grant no. GA-2014-127 to A.C.Z.). Chem. 273, 5858–5868 (1998). 27. Yuan, J., Adamski, R. & Chen, J. Focus on histone variant H2AX: to be or not to be. FEBS Lett. 584, 3717–3724 (2010). AUTHOR CONTRIBUTIONS 28. Luna, C., Yew, A. G. & Hsieh, A. H. Effects of angular frequency during clinor- A.C.Z. conceived the idea and designed the study, supervised the work, and wrote otation on mesenchymal stem cell morphology and migration. npj Microgravity 1, the manuscript. P.H. was involved in planning, study design validation, data 15007 (2015). collection, and contributed to writing the manuscript. A.L.R., R.L., N.C.D., E.J., and C.Z. 29. Xue, L., Li, Y. & Chen, J. Duration of simulated microgravity affects the differ- were involved in data collection and contributed to writing the manuscript. P.H. and entiation of mesenchymal stem cells. Mol. Med. Rep. 15, 3011–3018 (2017). S.C. performed the Engineering Validation Test. L.H., S.C., and L.S. were involved in 30. Akiyama, H. et al. Expression of PDGF-beta receptor, EGF receptor, and receptor planning during the early phase of this project, reviewed the manuscript, and adaptor protein Shc in rat osteoblasts during spaceﬂight. Mol. Cell. Biochem. 202, provided editorial comments. All authors reviewed and contributed to the ﬁnal 63–71 (1999). manuscript. 31. Chakraborty, N. et al. An integrated omics analysis: impact of microgravity on host response to lipopolysaccharide in vitro. BMC Genomics 15, 659 (2014). 32. Barretina, J. et al. CXCR4 and SDF-1 expression in B-cell chronic lymphocytic COMPETING INTERESTS leukemia and stage of the disease. Ann. Hematol. 82, 500–505 (2003). 33. Yonezawa, A., Hori, T., Sakaida, H. & Uchiyama, T. SDF-1 has costimulatory effects The authors declare no competing interests. on human T cells: possible involvement of MAPK (ERK2) activation. Microbiol. Immunol. 44, 135–141 (2000). 34. Meyer-Hoffert, U. et al. Th2- and to a lesser extent Th1-type cytokines upregulate ADDITIONAL INFORMATION the production of both CXC (IL-8 and gro-alpha) and CC (RANTES, eotaxin, Supplementary information is available for this paper at https://doi.org/10.1038/ eotaxin-2, MCP-3 and MCP-4) chemokines in human airway epithelial cells. Int. s41526-020-0106-z. Arch. Allergy Immunol. 131, 264–271 (2003). 35. Pype, J. L. et al. Expression of monocyte chemotactic protein (MCP)-1, MCP-2, and Correspondence and requests for materials should be addressed to A.C.Z. MCP-3 by human airway smooth-muscle cells. Modulation by corticosteroids and T-helper 2 cytokines. Am. J. Respir. Cell. Mol. Biol. 21, 528–536 (1999). Reprints and permission information is available at http://www.nature.com/ 36. Cervantes, J. L. & Hong, B. Y. Dysbiosis and immune dysregulation in outer space. reprints Int. Rev. Immunol. 35,67–82 (2016). 37. Bradley, J. H. et al. Simulated microgravity-mediated reversion of murine lym- Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims phoma immune evasion. Sci. Rep. 9, 14623 (2019). in published maps and institutional afﬁliations. 38. Shi, W. et al. Microgravity induces inhibition of osteoblastic differentiation and mineralization through abrogating primary cilia. Sci. Rep. 7, 1866 (2017). 39. Mitsuhara, T. et al. Simulated microgravity facilitates cell migration and neuro- protection after bone marrow stromal cell transplantation in spinal cord injury. Stem Cell Res. Ther. 4, 35 (2013). Open Access This article is licensed under a Creative Commons 40. Camberos, V. et al. Effects of spaceﬂight and simulated microgravity on YAP1 Attribution 4.0 International License, which permits use, sharing, expression in cardiovascular progenitors: implications for cell-based repair. Int. J. adaptation, distribution and reproduction in any medium or format, as long as you give Mol. Sci. 20, https://doi.org/10.3390/ijms20112742 (2019). appropriate credit to the original author(s) and the source, provide a link to the Creative 41. Baio, J. et al. Cardiovascular progenitor cells cultured aboard the International Commons license, and indicate if changes were made. The images or other third party Space Station exhibit altered developmental and functional properties. npj material in this article are included in the article’s Creative Commons license, unless Microgravity 4, 13 (2018). indicated otherwise in a credit line to the material. If material is not included in the 42. Mehta, S. K. et al. Reactivation of latent viruses is associated with increased article’s Creative Commons license and your intended use is not permitted by statutory plasma cytokines in astronauts. Cytokine 61, 205–209, https://doi.org/10.1016/j. regulation or exceeds the permitted use, you will need to obtain permission directly cyto.2012.09.019 (2013). from the copyright holder. To view a copy of this license, visit http://creativecommons. 43. Sanchez, L. et al. Enrichment of human ESC-derived multipotent mesenchymal org/licenses/by/4.0/. stem cells with immunosuppressive and anti-inﬂammatory properties capable to protect against experimental inﬂammatory bowel disease. Stem Cells 29, 251–262 (2011). © The Author(s) 2020 npj Microgravity (2020) 16 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA

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Feasibility, potency, and safety of growing human mesenchymal stem cells in space for clinical application

Feasibility, potency, and safety of growing human mesenchymal stem cells in space for clinical application

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Feasibility, potency, and safety of growing human mesenchymal stem cells in space for clinical application

Feasibility, potency, and safety of growing human mesenchymal stem cells in space for clinical application

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